Stool specimens from 152 hospitalized patients with diarrhea were analyzed for the presence of enterotoxigenic Bacteroides fragilis (ETBF) by a nested polymerase chain reaction (PCR) assay. ETBF gene sequences were directly detected in 14/152 (9.21%) stools of patients. The prevalence of ETBF in hospital-acquired diarrhea was statistically significant when compared to a prevalence of 2.3% in control subjects (P = 0.04). B. fragilis was cultured from 19.7% (30/152) patients with diarrhea; 4 of these isolates were enterotoxigenic. To determine whether colonization with B. fragilis is heterogeneous in nature, multiple colonies from 17 individual patients were analyzed for enterotoxin gene sequences and genotyped by arbitrarily primed PCR. Of these 17 patients, 13 harbored multiple strain types suggesting heterogeneity of colonization with both enterotoxigenic and non-enterotoxigenic strains. Identification of ETBF in the stools of 10 patients in the absence of a positive culture is likely due to the noted heterogeneity and suggests that detection of enterotoxin by PCR should be performed directly in the stool. These preliminary data indicate that ETBF may play a role in hospital-acquired diarrhea of unknown origin and suggest the need for further studies.