The prevalence of colonization with uropathogenic Escherichia coli and their reservoirs and routes of acquisition are incompletely defined. To help clarify these issues, polymerase chain reaction (PCR)-based strain typing assays were used to evaluate the fecal and vaginal E. coli flora of 11 volunteers. PCR detected the virulence genes papG, aer, and cnf significantly more frequently in mixed intestinal samples than in the corresponding predominant strains, evidence that traditional methods are suboptimal for detecting colonization with uropathogens. For strain typing, repetitive- element PCR was as discriminating as pulsed-field gel electrophoresis and O:H serotyping but more convenient. Molecular epidemiologic analysis of subjects' E. coli suggested emergence of occult uropathogenic strains from within the host's own intestinal flora, strain sharing between household members, and de novo acquisition of (unshared) uropathogenic strains. These methods should facilitate the studies needed to clarify the relative contributions of these three pathways to the pathogenesis of urinary tract infection.