To explore the potential protective role of urogenital lactobacilli against urinary tract infection (UTI), lactobacillus cultures were performed on stool and urine specimens and periurethral/vaginal swabs of febrile infants who were suspected of having UTI. Those infants diagnosed with UTI based on the results of the suprapubic urine cultures were allocated to the UTI group (n = 60), and those who had a simple viral illness with negative urine cultures were allocated to the control group (n = 31). Lactobacilli were anaerobically cultured in lactobacillus-specific DifcoTM Rogosa SL agar for 48 h at 37 degrees C and then counted. The lactobacillus colony counts for the stool and urine specimens and periurethral swabs from the UTI group were significantly lower than those for the control group (P < 0.05). The geometric means of stool, periurethra, and urine lactobacilli in the UTI group were significantly lower than those in the control group (P < 0.05). The colony count of the vaginal lactobacillus demonstrated an equivocal difference between the UTI and control group. In conclusion, this is the first prospective case-control study to demonstrate reduced lactobacillus urogenital colonization in infants with UTI. Our results support the view that less urogenital lactobacillus colonization may be a risk factor for UTI in infants even though there is an unclear possibility that the UTI itself could be the cause of the lower lactobacillus colonies.