A cytotoxic substance was identified in culture filtrates of several Escherichia coli strains, distinct from known heat-stable and heat-labile enterotoxins. It was toxic to Vero cells but not to Y-1 or CHO cells, and its effect on Vero cells differed from that of heat-labile enterotoxin (LT). The toxin was heat-labile and antigenically different from LT, with a molecular weight of 10,000 to 30,000. This toxin, named VT (toxic to Vero cells), was produced by some E. coli strains, including those from infants with diarrhea and cheese isolates. VT induced cytotoxic effects in Vero cells, with cell morphology changing and many cells floating in the medium. The effect progressed over time, with maximum titers observed after 4 days. VT was distinguished from LT in Vero cells, as LT caused enlarged, thick-walled cells with filamentous tendrils, while VT did not allow cell recovery. VT was neutralized by antiserum against H30 filtrate, except for H.I. 8 and E57. VT was heat-sensitive, with heating at 98°C for 15 min reducing its activity. VT was smaller than LT, with a molecular weight of 10,000 to 30,000 compared to 30,000 to 300,000. VT was cytotoxic, causing cell detachment, unlike LT, which stimulated cyclic adenosine 5'-monophosphate production. VT was found in 2 out of 66 food isolates, suggesting its potential role in diarrheal disease. Further studies are needed to determine VT's specific role in enteropathogenic disease.A cytotoxic substance was identified in culture filtrates of several Escherichia coli strains, distinct from known heat-stable and heat-labile enterotoxins. It was toxic to Vero cells but not to Y-1 or CHO cells, and its effect on Vero cells differed from that of heat-labile enterotoxin (LT). The toxin was heat-labile and antigenically different from LT, with a molecular weight of 10,000 to 30,000. This toxin, named VT (toxic to Vero cells), was produced by some E. coli strains, including those from infants with diarrhea and cheese isolates. VT induced cytotoxic effects in Vero cells, with cell morphology changing and many cells floating in the medium. The effect progressed over time, with maximum titers observed after 4 days. VT was distinguished from LT in Vero cells, as LT caused enlarged, thick-walled cells with filamentous tendrils, while VT did not allow cell recovery. VT was neutralized by antiserum against H30 filtrate, except for H.I. 8 and E57. VT was heat-sensitive, with heating at 98°C for 15 min reducing its activity. VT was smaller than LT, with a molecular weight of 10,000 to 30,000 compared to 30,000 to 300,000. VT was cytotoxic, causing cell detachment, unlike LT, which stimulated cyclic adenosine 5'-monophosphate production. VT was found in 2 out of 66 food isolates, suggesting its potential role in diarrheal disease. Further studies are needed to determine VT's specific role in enteropathogenic disease.