A cytotoxin inducing vacuolation in HEp-2 cells was detected in 19 (3.1%) of 618 stool specimens from children with diarrhea but in none of 135 from control children. Common enteric pathogens were found in only two (10.5%) of the 19 cytotoxin-positive stool specimens. The vacuoles induced by stool filtrates resembled those induced by the vacuolating toxin (VacA) of Helicobacter pylori. The vacuolating toxin was heat-labile and protease-sensitive, and it had an apparent molecular weight of >100,000 but was not neutralized by an antiserum to H. pylori VacA. Although proper prospective case-control studies are needed to definitely assess the etiologic association between the new vacuolating cytotoxin and diarrhea, the present study suggests that microrganisms of the gastrointestinal tract produce a Helicobacter-like vacuolating toxin and may be responsible for cases of childhood diarrhea whose etiology is currently considered unknown.