The pathophysiology of antibiotic-associated colitis was studied in rabbits with severe ileocolitis induced by oral administration of clindamycin. Cell-free, sterile filtrates of cecal contents of rabbits with clindamycin colitis contained a toxin that was lethal for mice and cytotoxic for HeLa-cell monolayers. The toxin was heat labile, was inactivated by pronase but not trypsin, and had a mol wt by gel filtration on Sephadex G-100 of 45,000. The toxin was neutralized by antiserum to Clostridium perfringens type E, but not by other clostridial antisera. The toxin also caused severe necrosis of rabbit rectal epithelium during 18-hr organ culture, which could be completely reversed by neutralization with C. perfringens type E antiserum. These studies indicate that clindamycin colitis in rabbits is caused by overgrowth of a clostridial species, which releases a heat-labile toxic protein of mol wt of 45,000 capable of necrosing colonic epithelial cells.