Eleanor (Geena Davis) and Frederick Little (Hugh Laurie) are intending to adopt a new family member. Their son George wants to go to the orphanage with them but has to go to school. They go to an orphanage where they meet and adopt an anthropomorphic mouse named Stuart (Michael J. Fox), whom they select over many other boys and girls up for adoption. Mrs. Keeper (the woman in charge) warns that humans should not be adopting mice. Stuart was helping the Littles trying to figure out who would be the right kid to adopt. Stuart knows almost everything about the other children, having been at the orphanage for a long time. Stuart wins them over by saying that he knows that nobody would want to adopt a mouse. However, Stuart is greeted coldly by their older son George (Jonathan Lipnicki), who refuses to acknowledge him as his brother. He was expecting a real boy and is disappointed as Stuart can't play any games with him. The family has a cat named Snowbell (Nathan Lane), who tries to eat Stuart. Family includes Uncle Crenshaw (Jeffrey Jones), Cousin Edgar (Brian Doyle-Murray), Grandpa Spencer (Harold Gould), Aunt Beatrice (Allyce Beasley), Aunt Tina (Connie Ray), Grandma Estelle (Estelle Getty), Uncle Stretch (Patrick Thomas O'Brien). That night, Snowbell visits Stuart and requests that he keep a low profile, so as to avoid getting seen by the other cats and damaging Snowbell's image (the alley cats would laugh forever if they heard that there was a mouse with a pet cat). Stuart's life with the Littles gets off to a bad start when George unknowingly throws him down the laundry chute with his pajamas and Eleanor puts him in the washing machine where he ends up submerged and swallows a lot of detergent making him really sick. He begins to feel like an outcast in the family when they bring him gifts and George snaps at them claiming that Stuart isn't his brother but simply a mouse. When Stuart admits he is lonely the Littles ask Mrs. Keeper (Julia Sweeney) to find Stuart's real parents. The Littles file an application at the orphanage. Stuart earns Snowbell's wrath when his friend, Monty the Mouth (Steve Zahn) visits for food. As Snowbell tries to keep him from seeing Stuart, Stuart makes a scene trying to get a cereal box from a cupboard. When he sees him and discovers he is a member of the family, Monty cannot contain himself. Enraged, Snowbell tries to kill Stuart, who narrowly escapes by fleeing into the basement. Here, Stuart learns that George keeps a playroom and the two interact and eventually, George comes to accept Stuart as his brother. Stuart also finds that George keeps a toy car just his size. Stuart finally bonds with George when they start playing together and plan to finish George's remote-controlled racing sailboat, Wasp, for an upcoming boat race on Conservatory Water in Central Park. Snowbell later goes with Monty to an alley to meet with Smokey (Chazz Palminteri), an alley cat mobster working for a mafia gang of other alley cats, who agrees with Snowbell to have Stuart removed from the household at his request. Stuart and George finish Wasp in time for the race, but on the day of the race, the controller is smashed by accident (by Stuart). Stuart pilots Wasp himself but ends up in a tussle with a larger boat piloted by George's bully, Anton (Miles Marsico) who is smashing all boats faster than his, as his own boat is larger and stronger. When he attempts to do the same thing to Stuart's boat, Stuart manages to bite into the line of his sail, rendering Anton's boat inoperable. Stuart manages to win the race, gaining George's respect. However, during the family celebration, the Littles are visited by a mouse couple, Camille (Jennifer Tilly) and Reginald Stout (Bruno Kirby), who claim to be Stuart's birth parents who sent him to the orphanage due to poverty. After discussion, Stuart reluctantly leaves with his real parents, taking the small car with him as a goodbye present from George and they go to their home, a small toy castle on a golf course. A few days later, Mrs. Keeper arrives at their house and reveals the truth to the Littles that Stuart's real birth parents died many years ago in an accident at a supermarket after a pile of canned food fell on them. Realizing that the Stouts are impostors, the Littles call the police Detective Allen (Jim Doughan) & Detective Sherman (Jon Polito), who begin a search operation. However, it is revealed that the Stouts were actually sent by Smokey, who had tricked them into posing as Stuart's parents and forced them to abduct Stuart from the Little household with the intention of having him brought over to the alley cat gang so they can kill Stuart. Realizing Stuart has been kidnapped, the family organizes a search party with "missing person" posters, using his photograph from the family photo. Fearing his involvement will be exposed and that he will be kicked out of the house, Snowbell informs Smokey about the news and Smokey settles on assassinating Stuart instead. Smokey calls a meeting with Reginald. However, he and Camille have grown to love Stuart like the Littles did and reveal the truth to him. In Central Park (on his way home), Stuart finds himself confronted by Smokey and his gang, who chase him through the park and into a sewer drain, where he manages to outrun them, but loses his car and luggage in the process. Stuart returns home, only to find that the Littles are already gone (they were all out looking for Stuart). Stuart enters the house where he meets Snowbell, who lies to him about the Littles enjoying themselves greatly since Stuart's departure. Snowbell shows him the family picture with Stuart's face cut out (the Littles had actually removed the picture to provide one for the Missing posters). Feeling unwanted, a heartbroken Stuart leaves and becomes discouraged. The Littles return home with no success in finding Stuart. Meanwhile, Smokey, Monty and the other alley cats manage to trace Stuart back to Central Park and bring Snowbell along for the hunt. Snowbell, having redeemed himself since he felt guilty over his selfishness, finds Stuart and rescues him from the cats while admitting that he lied. The cats catch up with them and Snowbell attempts to convince Smokey to call off the hit on Stuart but is refused. Instead, Smokey orders the cats to kill both Stuart and Snowbell. Although Snowbell defeats Monty and the other cats by snapping the tree branch on which they are standing, Smokey attempts to kill him upon discovering his betrayal, only for Stuart to intervene and save Snowbell by hitting Smokey in the face with another branch and knocking him out of the tree and into the water. He emerges but is immediately attacked by dogs. Stuart and Snowbell eventually return home, where Stuart happily reunites with the Little family, telling them that Snowbell actually found him and helped him on the way back, who has realized Stuart truly is family.
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