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A substance producing cytotoxicity in tissue culture was detected in stool specimens from all of four patients with pseudomembranous colitis due to antibiotics and in one of 54 with antibiotic-associated diarrhea. These stools also caused enterocolitis when injected intracecally into hamsters. On each occasion, cytotoxicity in tissue culture and enterocolitis in hamsters were neutralized by pretreatment with gas-gangrene antitoxin. The toxicity in both tissue cultures and hamsters could be reproduced with broth cultures of clostridia strains isolated from four of the five stools. These results suggest that toxin-producing clostridia are responsible for antibiotic-associated pseudomembranous colitis.