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In the beginning of the film, a boy named Eric Kirby (Trevor Morgan) and his soon-to-be stepfather, Ben Hildebrand, go parasailing near Isla Sorna with Dino-Soar Parasailing. But when the boat's crew are killed, Ben and Eric crash on the island. Scientists Alan Grant (Sam Neill) and Ellie Sattler (Laura Dern) have continued their paleontological careers but are working independently. Ellie is married and has two children; and Grant is still digging with his protégé, Billy Brennan (Alessandro Nivola). He is approached by Paul (William H. Macy) and Amanda Kirby (Téa Leoni), who say they are wealthy thrill-seekers who had charted a Beechcraft Super King Air 200 to tour Isla Sorna and want Grant to be a guide. Grant is reluctant at first, but he eventually agrees after the Kirbys promise to fund his dig.

Grant suspects that something is not quite right when the King Air 200 carrying him, Billy, the Kirbys', and a trio of mercenaries, Udesky (Michael Jeter), Cooper (John Diehl), and Nash (Bruce A. Young), lands. After Amanda uses a megaphone to try to call out to her son in the jungle, they are attacked by a Spinosaurus. They attempt to escape in the plane, leaving Cooper behind. Cooper tries to get them to stop the plane but is attacked on the runway by the Spinosaurus. They accidentally fly into the Spinosaurus and go down, fly through the forest and break apart when the Beechcraft Super King Air 200 hits the trees, stranding them on the island. The Spinosaurus attacks them again and tears off the front of the cockpit. Nash is pulled out of the plane and killed by the Spinosaurus. The plane falls to the ground and the Spinosaurus attempts to crush it, but Grant, Billy, Paul, Amanda, and Udesky escape. They then run into a Tyrannosaurus, which follows them into the forest. They soon run into the Spinosaurus again, and the two theropods engage in battle, with the Spinosaurus emerging victorious, by snapping the Tyrannosaur's neck. Grant learns that the Kirbys are actually searching for their son, Eric, who was stranded on the island along with Amanda's fiancé, Ben Hildebrand, in a parasailing accident 8 weeks beforehand. He decides to lead them to the coast, increasing their chances of getting rescued. Along the way, they discover the parasail, as well as Ben's skeletal remains. Billy salvages the parasail, and the Kirbys discover several nearby Velociraptor nests.

The group then explores the abandoned InGen compound, where they are attacked by a Velociraptor. As they flee, they are ambushed by the rest of the raptor pack, and Udesky gets separated from the others and is killed. When Grant becomes separated from the group, he is rescued from several raptors by the young Eric Kirby (Trevor Morgan), who has been living on the island for eight weeks in an abandoned supply truck. When the group reunites, they are attacked again by the Spinosaurus. After they find shelter in another building, Grant finds out that Billy has stolen two Velociraptor eggs in the hope of selling them upon their return to the mainland and funding the dig; this provoked the earlier attack. Appalled, Grant tells Billy, "As far as I'm concerned, you're no better than the people that built this place."

To reach a boat docked in a nearby river, the group must pass through a massive aviary dome, where they are attacked by numerous Pteranodons. Using the parasail he salvaged, Billy redeems himself by rescuing Eric from a nest of Pteranodon infants, but is attacked and overwhelmed by several adults, and the rest of the group assumes he is dead. Amanda and Eric fail to lock the Pteranodons inside as the group escapes the dome. Grant and the Kirbys board the boat, and while floating down the river, they hear a phone ringing. This leads them to a dung pile containing a satellite phone the Spinosaurus had eaten from the plane, which they are forced to sift through in order to recover the phone. Ceratosaurus appears briefly but doesn't attack, due to the smell of the dung on the humans. Grant attempts to contact Ellie via the satellite phone, but only communicates "The river- Site B! The river!" as they are again attacked by the Spinosaurus while floating down river. Paul is briefly thought to have been killed in the attack, but manages to survive, and the Spinosaurus finally flees after it is shot with a flare gun.

The group is close to the shore when the raptors reappear, wanting their eggs back. The raptors pause when Grant imitates a Velociraptor call on a special pipe he had formed at the start of the film, based on a raptor's skull. Suddenly, a helicopter can be heard overhead. The eggs are given back to the raptors, and they retreat. The group arrives at the beach to see a United States Marine Corps detachment sent by Ellie's diplomat husband. As they board a helicopter, Grant finds Billy, who was injured during the Pteranodon attack but is still alive. As the helicopter flies off, the survivors see three Pteranodons flying off into the distance. Eric asks Grant where they might be going, and Grant speculates that they are searching for new nesting grounds. The trio of Pteranodon are seen flying off in the clouds.

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