In Pixar's Cars, Lightning McQueen accidentally leaves Interstate 40 while driving to California.
The mistake occurs during the opening journey to the Piston Cup tiebreaker in California. Lightning becomes separated from his transport truck, Mack, and takes the wrong turn while trying to catch up. The detour eventually leads him toward Radiator Springs, where his reckless driving damages the town's road.
Interstate 40 is a real east-west highway that crosses the United States, including areas near historic Route 66. Cars uses the highway and the old road as part of its contrast between fast modern travel and slower roadside communities.
Route 66 is therefore closely connected to the setting, but it is not the highway Lightning leaves in this scene. The film's highway sequence is also the event that sets up his court-ordered work in Radiator Springs.