BACKGROUND E. coli strains producing specific virulence factors are frequently cited as causes of pediatric diarrhea in developing areas, although many well children from the same areas are colonized with these organisms. The role of these Escherichia coli in day-care center (DCC)-associated diarrhea in the United States has not been evaluated. METHODS A cohort of 112 DCC attendees from 5 DCC in urban New Orleans were followed longitudinally with demographic data, biweekly routine stool samples and additional stool samples with episodes of diarrhea. E. coli isolates were routinely saved; diarrhea stool samples were tested to detect enterotoxigenic, enterohemorrhagic and enteroadherent strains; and the prevalence of these E. coli in children with and without diarrhea was investigated. RESULTS During 225 child months of observation 21 episodes of diarrhea were documented and microbiologic data were available for 18. HEp-2 cell enteroadherent E. coli [mostly enteroaggregative (EAggEC) pattern] were identified in 6 of 18 (33.3%) diarrhea cases vs. 6 of 36 (16.6%) age-matched controls. However, the prevalence of EAggEC was very DCC-specific, with EAggEC found in 12 of 22 routine specimens from a DCC with recent EAggEC-related diarrhea vs. 0 of 11 routine specimens from age-matched children in another DCC without EAggEC-related diarrhea (P=0.002). Enterotoxigenic E. coli were uncommon in both ill and well children, and no enterohemorrhagic E. coli were detected. CONCLUSIONS EAggEC were commonly isolated from children with and without diarrhea in certain DCC settings, although we cannot determine whether these strains caused diarrhea. Diarrhea-producing E. coli were not associated with diarrhea in this DCC population.