Two toxins were isolated from a toxigenic strain of Clostridium difficile. The toxins were purified by gel filtration and ion-exchange column chromatography to homogeneity as judged from polyacrylamide gel electrophoresis and were designated D-1 and D-2. Toxin D-1 was lethal for mice, increased vascular permeability, and induced fluid accumulation in ligated rabbit ileal loops, and toxin D-2 displayed cytotoxicity in HeLa cells with a minimum of 1 pg of toxin. The molecular weights of toxins D-1 and D-2, as estimated by gel filtration, ranged from 550,000 to 600,000 and from 450,000 to 500,000, respectively. These toxins were heat labile and inactivated by pronase and trypsin. Amino acid analyses of both toxins showed them to be of relatively similar composition. Antisera prepared against purified toxin D-1 neutralized all of the biologic activities of toxin D-1, whereas it did not affect any of the biologic activities of toxin D-2. A sensitive latex-agglutination immunoassay was developed for screening for C. difficile toxin D-1 in patients with pseudomembranous colitis.