Since diarrheagenic E. coli are not identified by common clinical laboratory techniques, we hypothesized that these organisms might be an unrecognized cause of enteritis in children in the U.S. 1327 children with acute gastroenteritis were identified prospectively by active surveillance in the Emergency Department (ED) and the inpatient units at Cincinnati Children's Hospital Medical Center. Stool samples were evaluated for diarrheagenic E. coli using a panel of DNA probes and adherence pattern to HEp-2 cells. Stool samples from a reference group of 555 well children were studied for comparison. Gene probe studies, but not HEp-2 cell adherence, demonstrated that enteroaggregative, diffusely adherent and enteropathogenic E. coli were associated with clinical illness. Each was isolated significantly more often from study subjects in the ED than controls. In children <1 year of age, enteroaggregative E. coli were isolated significantly more often from both inpatients (4.7%, Odds Ratio = 3.4, 95% confidence intervals 1.3-9.1, p <0.03) and ED patients (10.0%, Odds Ratio = 7.2, 95% confidence intervals 2.9-18.2, p <0.001) than from well children (1.4%). Diarrheagenic E. coli, especially enteroaggregative E. coli, may be an important, unrecognized cause of childhood diarrhea in the U.S.