April O'Neil is a news reporter for Channel 6 Eyewitness News in New York City investigating a crime wave by a clan of thieves called the Foot Clan. After her latest lead; a dock worker, refuses to give her information, she returns to the dock at night only to witness the Foot raiding the cargo containers. They are attacked by an unseen vigilante, which April fails to capture on her phone's camera but notices a symbol left behind. However, April's supervisor Bernadette Thompson and her co-workers are oblivious to her beliefs of the vigilante's existence. At a charity event she is covering for the company Sacks Industries, she expresses her gratitude to her father's lab partner before he died, Eric Sacks.
Frustrated by the vigilante, the Foot Clan's leader Shredder has them hold a subway station of people hostage in order to draw him out. April goes to the scene, for evidence but ends up becoming a hostage herself. The lights go out, and four figures appear to face the clan, taking them out and freeing the hostages. April tracks them to a rooftop, and manages to take their picture, but she passes out when she is confronted with four anthropomorphic mutant turtles. When she comes to, she finds that they had wiped her phone and advise her not to tell anyone of them before leaving. As they leave, she manages to hear Raphael and Leonardo's names. She returns to her apartment and remembers a project her father was working on in 1999; "Project Renaissance" involving four turtles named Leonardo, Donatello, Michaelangelo, Raphael, and a rat she called Splinter. Unable to convince Bernadette of the turtles' existence, April is dismissed. April has her coworker Vern Fenwick drive her out to Eric Sacks' estate where she confides in him about what she has discovered. Eric believes her and reveals that he hopes they can unlock the secrets of a mutagen they had been experimenting with to cure disease in the populace thought lost in the fire.
Punishing them for venturing to the surface, Splinter goads Mikey into revealing they met April, and he demands to have her meet them. When they bring her to their sewer lair, Splinter explains that April had saved them all from the fire and freed them into the sewers. The mutagen being experimented upon them caused the five of them to grow into more humanoid attributes, and Splinter took on the role of their father, using April's father as an example. After finding a book on Ninjitsu in a storm drain, he proceeded to teach himself, then the turtles in the fighting style. April reveals that she told Sacks, whom Splinter says betrayed her father and killed him, just then, the Foot attacks. They fight valiantly until Shredder arrives, who defeats Splinter as he is trying to keep his turtles from entering a fight with Shredder, knowing Shredder is a far superior warrior than the four mutants. A series of explosives bury Raph in rubble while the other turtles are captured. April comes out of hiding and she and Raph decide they need to save the others. Finally convincing Vern of the turtles' existence, he drives them to Sacks' estate and break in as the others are being drained of their blood in order to create an antidote to a deadly virus that Sacks hopes to flood New York with, in order to become rich from all the people seeking his cure. Raph is beaten down by Shredder while April and Vern free the others by injecting them with adrenaline. They escape the compound in pursuit of Sacks, but are followed by heavy machines and trucks. The group manages to survive the onslaught through teamwork, though Vern's van is destroyed.
In the city, Sacks and Shredder plant a device on a high radio tower to flood the city. April and Vern face Sacks in the lab, while the turtles fight Shredder on the roof. During the fight, the support for the tower collapses and the turtles try to hold it up and keep it from falling and infecting the city. April arrives and confronts Shredder with the mutagen, in the struggle, the tower collapses and the turtles pull April onto it with them as Shredder falls to the streets. Believing they're about to be destroyed, the turtles confess their innermost secrets, Raph 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 has a new loaner car from the news station, he tries and fails once again to ask April out on a date. The turtles appear in a special modified "Turtle Van" and Mikey accidentally blows up Vern's new car with a rocket. As the police respond to the explosion, the turtles leave, but not before Mikey tries to serenade April with "Happy Together".
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