An outbreak of food-borne infection with enterotoxigenic Escherichia coli (ETEC) O6: H16 producing heat-labile enterotoxin and heat-stable enterotoxin occurred in Miyazaki Prefecture, Japan, in September 1999. During this outbreak, 211 out of 1, 439 people examined (14.7%) were affected. The major clinical symptoms were diarrhea and abdominal pain. However, data from epidemiological surveillance of infectious diseases in Miyazaki Prefecture showed that ten sporadic diarrhea cases caused by ETEC O6: H16 occurred diffusely in the prefecture for some weeks before and after this outbreak. In order to clarify the relationship between the outbreak and the sporadic cases, the isolates from each case were analyzed genetically by pulsed-field gel electrophoresis method (PFGE). As a result, the PFGE patterns of these isolates, except for one sample from a sporadic case, were observed to be the same, after cleavage of DNA with restriction enzymes Xbal, Notl and ApaI. These results suggest that the outbreak and the sporadic cases may have represented a diffuse outbreak initiated by contaminated foods appearing on the market in Miyazaki Prefecture.