Proteus mirabilis, an etiologic agent of complicated urinary tract infections, expresses mannose-resistant Proteus-like (MR/P) fimbriae whose expression is phase variable. Here we examine the role of these fimbriae in biofilm formation and colonization of the urinary tract. The majority of wild-type P. mirabilis cells in transurethrally infected mice produced MR/P fimbriae. Mutants that were phase-locked for either constitutive expression (MR/P ON) or the inability to express MR/P fimbriae (MR/P OFF) were phenotypically distinct and swarmed at different rates. The number of P. mirabilis cells adhering to bladder tissue did not appear to be affected by MR/P fimbriation. However, the pattern of adherence to the bladder surface was strikingly different. MR/P OFF colonized the lamina propria underlying exfoliated uroepithelium, while MR/P ON colonized the luminal surfaces of bladder umbrella cells and not the exfoliated regions. Wild-type P. mirabilis was usually found colonizing intact uroepithelium, but it occasionally adhered to exfoliated areas. MR/P ON formed significantly more biofilm than either P. mirabilis HI4320 (P = 0.03) or MR/P OFF (P = 0.05). MR/P OFF was able to form a biofilm similar to that of the wild type. MR/P ON formed a three-dimensional biofilm structure as early as 18 h after the initiation of the biofilm, while MR/P OFF and the wild type did not. After 7 days, however, P. mirabilis HI4320 formed a 65-mum-thick biofilm, while the thickest MR/P ON and MR/P OFF biofilms were only 12 mum thick. We concluded that MR/P fimbriae are expressed by most P. mirabilis cells infecting the urinary tract, dictate the localization of bacteria in the bladder, and contribute to biofilm formation.