Stools were tested for the presence of a cytopathic toxin that is neutralized by gas gangrene antitoxin or Clostridium sordellii antitoxin. The test was positive in specimens from 51 (96%) of 53 patients with antibiotic-associated pseudomembranous colitis and five (2%) of 248 individuals without gastrointestinal complications of antimicrobial usage. Seven types of tissue cultures were tested, and all proved susceptible to this toxin. Those that appeared to be the most susceptible on the basis of titers of toxin in stools were primary human amnion cells, WI-38 cells, baby hamster kidney cells, and mouse fibroblasts. Storage of specimens at -70 C, 6 C, 28 C, and 37 C for five days before testing resulted in decrease in toxin titers that correlated with increasing temperatures. Studies of various C. sordellii antitoxin preparations indicated considerable variation in potency. Recommendations for performing the tissue culture assay for the toxin found in stools of patients with antibiotic-associated colitis are provided on the basis of these observations.