In 2009, a plague caused by a infected vampire bat has transformed most of the world's population into vampires. This event formed a world completely dominated by vampires. As vampires are incapable of aging or dying, but are unprotected against the sunlight or any ultraviolet light, the entire vampire world is active at night, and various technological and architecture advances are made to adjust the populated areas to vampires' night cycle (such as UV protective cars, underground subways and paths across the cities) and UV warnings.
Human numbers quickly dwindle and the need for blood becomes desperate, while a global war rages between the surviving humans and vampires, and the vampires emerge victorious. When deprived of blood for extended periods, vampires degenerate into "subsiders," psychotic bat-like creatures which lose their memories and independent thought and left with the basic need for blood. Humans are captured and harvested in laboratory farms while scientists research a synthetic blood substitute to satisfy vampires' blood hunger across the world.
In 2019, Edward Dalton (Ethan Hawke) is the head hematologist for the pharmaceutical company Bromley Marks, the largest supplier of human blood in the United States. Along with fellow hematologist Christopher Caruso (Vince Colosimo), Dalton is in the process of developing a substitute to bolster dwindling blood supplies and to create a substance that can replace blood and be generated. The need is underscored after Dalton's boss, company owner Charles Bromley (Sam Neill), reveals that the estimated human population is down to 5%, and national blood supplies will not last more than a month. Faced with this knowledge, Edward and Chris carry out a hasty clinical trial of the latest revision on a volunteering soldier, which is a spectacularly gruesome failure when the soldier explodes from a painful side-effect.
On the drive home, Dalton becomes momentarily distracted by lighting a cigarette and seeing his ears becoming longer (first sign of becoming a subsider) and accidentally runs another vehicle off the road. When he checks on the occupants of the other vehicle, he is shot in the arm by a crossbow shot, and quickly discovers they are humans. With police approaching, Dalton convinces the humans to hide in his vehicle and activates the UV protector which tints the glass to hide them, and then tells police that the occupants of the other vehicle fled. The humans then leave, but not before their leader, Audrey (Claudia Karvan), learns Edward's name and occupation from the ID badge on his jacket.
At home, Edward is surprised by his estranged brother Frankie (Michael Dorman), a soldier in the human-hunting Vampire Army who has returned for Edward's 35-th birthday. Frankie's gift of a bottle of pure human blood re-ignites a long-standing argument over Edward's sympathies towards humans, his refusal to drink human blood (he drinks animal blood as a substitute), and his resentment towards Frankie for turning him into a vampire. The argument is cut short when a subsider invades Edwards house, forcing the brothers to team up and kill it, and he discovers that the subsider is actually a gardener that went missing weeks ago.
The following morning, Audrey arrives at Edward's home, giving him a note with instructions for a meet before departing. After some consideration, Dalton goes to the meeting location during the day in his UV protected Chrysler 300C and hides in the shades of a tree, and is introduced to Lionel "Elvis" Cormac (Willem Dafoe), another human, but an Army SUV arrives with Frankie, who followed Edward and intends to capture both Cormac and Audrey. Audrey knocks Frankie unconscious and the three make their escape, fleeing from approaching soldiers in Edward's car (since Cormac's orange 1965 Ford Mustang is destroyed with bullets). Once they escape, Cormac drives to the edge of a river, where he reveals that he was once a vampire and a professional mechanic, and one of the pioneers in creating a UV protection cover for windows and cameras, and lived close to Edward, but was cured when a car crash ejected him from his sun-proof vehicle into the river during daylight hours. He asks Dalton to help find a way to recreate the cure safely; Dalton agrees.
That night, Ed arrives with Elvis and Audrey at an old vineyard. Ed meets with the other human survivors, as well as Senator Turner (Jay Laga'aia), a vampire who secretly helps the colony to find a cure. While a convoy of humans is heading to Audrey's group, they are ambushed by vampire soldiers, and captured. One soldier finds a radio and the soldiers track the position of the vineyard, forcing the humans there to flee. Audrey, Elvis and Ed stay behind so Ed can be turned back. After some experimentation, Edward is able to reverse the vampire effect, curing himself, and feels sunlight for the first time in a decade. They depart in Cormac's 1978 Pontiac Trans Am.
One of the captured convoy inhabitants is Alison (Isabel Lucas), who wakes up in Charles' office. She is revealed to be his daughter, who ran away after the outbreak of the plague, not accepting her father as a vampire. Charles, wanting to have his daughter back, has Frankie forcibly turn her. She refuses to drink human blood, feeding on her own instead - and thus begins to turn into a subsider. She is rounded up with a group of the creatures in a chain gang and dragged into sunlight to burn to death. Witnessing Alison's death upsets Frankie and he seeks out his brother, having an epiphany. Meanwhile, Bromley Marks has exhausted their reserves of blood, and the army arrives in the cities, destroying subsiders and vampires in poverty to control the population.
Edward, Elvis, and Audrey break into Chris' home and ask him to help spread the cure. However, Chris has finally discovered a viable blood substitute and does not want there to be a cure, since the substitute will make him a rich and powerful man, and admits that he always hated Edward and was always in his shadow and now wants to take over Edward's place. He calls in vampire soldiers, who capture Audrey while Elvis and Edward escape into hiding. They are found by Frankie, who agrees to help but is gradually becoming a subsider. He attacks Elvis, but drinking his blood turns him into a human again, and they discover that feeding on a former vampire is another method for vampires to turn back to humans.
Edward, trying to save Audrey, turns himself in. Charles reveals that he is not interested in the cure, and reveals that before being a vampire, he suffered from incurable cancer, and after he became a vampire, he turned the Bromley Marks into a powerful company. He admits that he likes being immortal and that he will use the substitute world-wide to create a monopoly over the market, and that the remaining human blood will become very expensive, since "there are always people who are willing to pay extra for the real thing". He then bites Edward to turn him into a vampire, but he shifts back into a human.
Edward uses the now-human Charles to cure a group of soldiers starting to experience subsider aggression (the soldiers end up ripping Charles up alive). Edward and Audrey, cornered by more soldiers, are rescued when Frankie sacrifices himself. This sparks a feeding frenzy that leaves all of the soldiers dead or cured. They are confronted by Chris, who kills the few remaining cured soldiers to hide the evidence of the cure. Just as Chris points his gun at him, Elvis arrives and shoots him in the chest with a crossbow. Edward looks sadly to his dead brother, and Audrey, Edward and Elvis turn back and watch the sun rise on the city.
The three survivors then drive away in Cormac's Pontiac in the horizon, with a voice-over by Edward stating that they have a cure and can change others back. Just before the screen turns to black however, a subsider is seen flying in the sky howling, revealing that it is maybe too late.
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