Hamsters treated with vancomycin developed enterocolitis significantly more often in a conventional animal room than in a room designed to prevent cross-infection with Clostridium difficile. In the conventional room C. difficile was isolated from cages, food racks, floors, buckets, the hands of caretakers, and the stools of animals with enterocolitis but not from untreated hamsters, air, or food from freshly opened bags. C. difficile was not isolated from environmental sources in the clean room. It was not possible to determine which of the sources of the organism was most important in its spread. Cross-infection with C. difficile may be important in the pathogenesis of antibiotic-associated enterocolitis in hamster colonies.