Mrs. Brisby, a widowed mouse, lives in a cinder block with her children on the Fitzgibbon farm. She is preparing to move her family out of the field they live in as plowing time approaches, however her son Timothy has fallen ill, and moving him could prove fatal. Mrs. Brisby visits The Great Owl, a wise creature who advises her to visit a mysterious group of rats who live beneath a rose bush on the farm. Upon visiting the rats, Brisby meets Nicodemus, the wise and mystical leader of the rats, and Justin, a friendly rat who immediately becomes attached to Mrs. Brisby. While there, she learns that her late husband, Mr. Jonathon Brisby, along with the rats, was a part of a series of experiments at a place known only as N.I.M.H. (revealed earlier in the story as the National Institute of Mental Health). The experiments performed on the mice and rats there boosted their intelligence, allowing them to read without being taught and to understand things such as complex mechanics and electricity. The rats and Mr. Brisby escaped from N.I.M.H. and came to live on the Fitzgibbon farm. The rats created a home for themselves under Mrs. Fitzgibbon's rose bush, creating an elaborate habitation of beautiful chambers, elevators, and Christmas lights. However, the rats are unhappy in their dependence on the humans, who they are stealing electricity from, and have concocted a plan to leave the farm and live independently. Because of her husband's prior relationship with the rats, they agree to help Mrs. Brisby move her home out of the path of the plow. However, the evil Jenner and his unwilling accomplice Sullivan, who wish to remain beneath the rose bush, yet plot to kill Nicodemus during the move.

Charlie B. Barkin (Burt Reynolds), a rascally German Shepherd with a shady past, breaks out of the New Orleans Dog Pound with the help of his faithful friend Itchy (Dom De Luise), a nervously hyperactive dachshund. He then makes tracks to reunite with his gambling casino partner Carface Malone (Vic Tayback), a shifty pitbull who has planned a dastardly, and potentially deadly, double cross. Suddenly, Charlie finds himself at the Pearly Gates, face to face with the Heavenly Whippet (Melba Moore). Charlie weasels his way back to earth and reunites with Itchy. He plots his revenge against Carface and, along the way, acquires help from a little girl named Anne-Marie (who can talk to animals). After a series of fiendish schemes, close scrapes and unexpected adventures, both Charlie and Anne Marie find their lives at stake. Only one can be saved and the outcome is in Charlie's paws...

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