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An epidemic of enterohemorrhagic colitis caused by Escherichia coli O157:H7 (EHEC-O157) occurred in a nursery school in a rural area of Japan in September 1996. The EHEC-O157 were isolated both from patients and houseflies collected at the school. The flies were suspected to be mechanical vectors of the pathogen. Feeding experiments of EHEC-O157 to houseflies showed that the ingested bacteria were harbored in the intestine of flies and continued to be excreted at least for 3 days after feeding. Scanning electron microscopy showed that a large number of EHEC-O157 adhered to the surface of the housefly mouthparts and actively proliferated in the minute spaces of the labellum. Food masses containing EHEC-O157 in the fly intestine were completely surrounded by a peritrophic membrane during digestion and discharged rapidly. The persistence of bacteria in the intestine and feces is mainly a result of proliferation in the mouthparts and accumulation in the crop. Our results strongly suggest that houseflies are not simple mechanical vectors of EHEC. The epidemiologic potential of houseflies to disseminate EHEC-O157 may be greater than initially suspected.