Species-specific colonization of rabbit intestine by RDEC-1 Escherichia coli is an accepted animal model for bacterial mucosal adherence. To determine whether RDEC-1 pili are functional as adherence factors for this organism, we grew the organism under conditions that promoted pilus expression; we isolated the pili, documented their purity, and compared their mucosal adherence properties with those of whole organisms using an indirect immunofluorescence technique. Frozen sections of rabbit, rat, guinea pig, and human small intestine were incubated with either piliated or nonpiliated RDEC-1 organisms or purified RDEC-1 pili and observed for the distribution and intensity (0–4+) of fluorescence. Piliated RDEC-1 organisms fluoresced brightly (4+) and were distributed along the entire mucosal surface of the rabbit ileum. Only a few nonpiliated RDEC-1 attached to rabbit ileum, and they were randomly scattered across the entire section of tissue. Rabbit ileum overlain with pure RDEC-1 pili showed a specific, d-mannose resistant (2–3+) fluorescence on the mucosal surface from the crypts to the villus tips. No fluorescence was seen on the guinea pig, rat, or human mucosal surface overlain with RDEC-1 pili. Purified RDEC-1 pili adhere to the rabbit intestinal mucosa in a species-specific manner and with the same distribution as whole piliated organisms. The data suggest that RDEC-1 produce pili (distinct from type 1 pili) that determine the specificity of the mucosal adherence of RDEC-1 to rabbit ileum.