- “You gettin' big, Jeff. Boy, what you been eatin'?”
- —Agent J
In the beginning of the movie, Jay and Tee walk down the street when they come across the flower on top of Jeff's head in the subway system. Jay starts talking friendly to Jeff while Tee kicks and pulls the flower, despite Jay repeatedly advising him not to. Jeff is angered and bursts out of the ground, taking Tee with. Jeff then slams Tee into the ground and returns underground. Jeff then proceeds to burst his tail out of the ground and hits Jay with it. Jay then jumps into the hole that Jeff burrowed through and finds himself on top of Jeff as he slithers through the subway. Jay proceeds to inject Jeff with a type of tranquilizer that has no effect on Jeff. Jay is then launched forward through the back window of a train. Jay tells everyone on the train to move to the front wagon, but is ignored. However, as soon as Jeff starts eating up the train, the passengers starts moving forward. Jay then threatens Jeff with a gun, and Jeff, now feeling guilty for what he just had done, stops eating the train, and by this time the tranquilizer finally takes effect and Jeff falls to sleep. Afterwards, Jay leaves the subway and joins Tee in a diner (where he promptly neuralyzes him after he came too close to exposing his true status as an agent while having an emotional breakdown over the prior events).
Before Jay heads off to restore his former partner Agent K's memories, Jeff was mentioned when J went to talk with Agent Z, which he before sending Jay on his next mission, congratulates him for bringing Jeff back to his senses and states that he knew Jeff since he was tiny, implying that Jeff was part of MIB since he was an infant.
Near the end of the movie, Kay, Jay, and Laura are chased by Serleena, by ship, into the subway system. Here, Jay gets close to Jeff and drops on the ground and Serleena is eaten. As Laura leaves Earth, Jeff comes to the top of the building, roaring in pain, much to Jay's Fury as he Shouts at him telling him that he was not in the mood for him and ordered him to get back into the subway as Serleena has survived and is now taking over his body. Just as Serleena finally grows into size, she burst Jeff to his death in pieces, much to Jay, Kay, and Laura's shock.