To assess the role of antecedent rectal and urethral colonization in the pathogenesis of catheter-associated urinary tract infections, we prospectively studied 64 patients, obtaining urine, rectal, and urethral cultures on study entry, two days later, and every four days thereafter. Of 55 patients who remained catheterized for at least three days, urethral colonization with the same microorganism present in bladder urine was observed in the sampling period preceding the onset of bacteriuria in 12 of 18 infections in women and five of 17 in men. Rectal colonization with the infecting strain preceded bacteriuria in 14 of 18 infections in women and five of 17 in men. The identity of the urinary, urethral, and rectal isolates was confirmed by speciation, serotyping, antibiograms, and biotyping. We conclude that rectal and periurethral colonization often precedes catheter-associated bacteriuria, especially in women, and that effective preventive measures aimed at transurethral infection should be developed.