April O'Neil, a local reporter for Channel 6 Eyewitness News in New York City, investigates a crime wave by a group of criminals called the Foot Clan. At a dock at night, she sees the Foot raiding cargo containers. After an unseen vigilante attacks the thieves, April notices a symbol left behind. April's supervisor Bernadette Thompson (Whoopi Goldberg) and her coworkers are oblivious to her story. Later while covering a charity event thrown by Sacks Industries, April expresses gratitude to the company's CEO Eric Sacks, who was her late father's lab partner.
Frustrated by the vigilante, the Foot Clan's leader Shredder has the Foot Soldiers take hostages at a subway station in order to draw him out. April, at the scene, becomes a hostage herself. Four mysterious figures arrive, take out the Clan, and free the hostages. April follows them to a rooftop and is shocked to see that the vigilantes are anthropomorphic mutant turtles, causing her to pass out. When she regains consciousness, they advise her not to tell anyone of them. As they leave, April hears Raphael and Leonardo's names.
April returns to her apartment and remembers "Project Renaissance", her father's science experiment, which involved four turtles named Leonardo, Donatello, Michelangelo, Raphael, and a mutated rat called Splinter. Unable to convince Bernadette of the Turtles' existence, April is dismissed. Her coworker Vern Fenwick drives her to Sacks' estate where she confides in him about her discovery. Sacks believes her and reveals that he and April's father had been experimenting on a mutagen created to cure disease, which was thought lost in the fire that killed her dad.
At Splinter's behest, the Turtles bring April to their sewer lair. Splinter explains April had saved them all from the fire and freed them into the sewers. The mutagen caused the five of them to grow and develop humanoid attributes. Splinter took on the role of their father, using April's father as an example. After finding a book on Ninjutsu in a storm drain, he proceeded to teach himself, then the Turtles, in the fighting style. When April reveals she told Sacks about her discovery of the Turtles, Splinter informs her that Sacks turned on her father and killed him.
Then, Shredder and the Foot Soldiers attack the lair, defeating Splinter and incapacitating Raphael while the other Turtles are captured. April comes out of hiding and she and Raphael plan to save the others. At Sacks' estate, he has the Turtles' blood drained in order to create an antidote to a deadly virus that Sacks hopes to flood New York with, believing he will become rich from people seeking his cure. Raphael, April, and Vern storm the estate and free the other Turtles. The group then escapes the compound in pursuit of Sacks.
On a radio tower in the city, Sacks and Shredder plant a device that will flood the city with the virus while Sacks is preparing to convert the mutagen to healing factor. April and Vern subdue Sacks in the lab, while the Turtles are battling Shredder on the roof. During the fight, the tower's support beams collapse. As the turtles try to keep it from falling and infecting the city, April confronts Shredder with the mutagen. In the struggle, the tower collapses and the Turtles pull April onto it with them, while Shredder falls to the street and is confronted by police. Believing they are about to die, the Turtles confess their secrets, while Raphael gives an impassioned speech of his love for his brothers before they land harmlessly on the street. They vanish before the humans find them and return to the sewers, where they give Splinter the mutagen and he begins to recover.
Sometime later, April meets with Vern, who tries and fails to ask her on a date. The Turtles appear in a special modified "Turtle Van", and Michelangelo accidentally blows up Vern's new car with a rocket. As police respond to the explosion, the Turtles leave, but not before Mikey tries to serenade April with "Happy Together" by the band The Turtles, much to his brothers' annoyance and April's amusement.
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