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To investigate the etiologic role of Clostridium difficile in childhood acute diarrhea, stool specimens from 618 children with diarrhea and 135 controls without enteric symptoms were examined by cell culture assay for the presence of free toxin B. This toxin was found in 4.2% of the fecal specimens examined without finding a significant difference between cases and controls, suggesting no causal relationship between diarrhea and the presence of free C. difficile toxin B. C. difficile strains isolated from toxin B-positive specimens were characterized by cytotoxin and enterotoxin production and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EDTA-extracted proteins. All but two isolates produced toxin B and toxin A and the remaining were negative for both toxins. Polyacrylamide gel electrophoresis analysis showed eight electrophoretic types, none of them was clearly related with the cases of diarrhea. The majority of isolates from children with diarrhea did not belong to types previously observed in adults with pseudomembranous colitis or antibiotic-associated diarrhea. This study provides additional evidence that C. difficile is not involved in the etiology of childhood diarrhea.