Tissue cultures were performed on stools from 189 patients to detect a cytopathic toxin which is neutralized by Clostridium sordellii antitoxin. Specimens satisfying these criteria were considered positive in the tissue culture assay. Stools from 26 of 27 patients with antibiotic-associated pseudomembranous colitis were positive and 16 of these specimens showed toxin titers of 10⁻³ dilutions or greater. The tissue culture assay was positive with specimens from 9 of 63 patients with antibiotic-associated diarrhea without documented pseudomembrane formation. Stools from patients with neonatal necrotizing enterocolitis, ulcerative colitis, and healthy controls were uniformly negative in this assay. Cultures were performed on stools from 38 patients with antibiotic-associated diarrhea or colitis to detect clostridia which produce a cytopathic toxin in vitro. Clostridium difficile was recovered from 6 of 8 specimens which were positive in the tissue culture assay and 5 of 30 which were negative in this assay. C. sordellii was recovered in a single specimen. One hundred and nine clostridia strains were tested in the tissue culture assay and C. difficile was the only species which produced a cytopathic toxin. All strains of this organism were positive in the tissue culture assay and, in each instance, cytotoxicity was neutralized by C. sordellii antitoxin. These results indicate that C. difficile is the major cause of antibiotic-associated pseudomembranous colitis and offer an explanation for previous studies showing that the cytotoxin of stools from these patients is neutralized by C. sordellii antitoxin.