We examined the activities of Clostridium difficile toxin preparations given intragastrically to hamsters, mice, and rats. The culture filtrate from a highly toxigenic strain of C. difficile caused hemorrhage and accumulation of fluid in the small intestine and cecum, diarrhea, and death in hamsters and mice. In rats, the culture filtrate caused only a small amount of fluid accumulation and slight hemorrhage along the small intestine. When toxin A was removed from the culture filtrate, the filtrate lost its activity. Preparations of homogeneous toxin A caused a response similar to that observed after the administration of culture filtrate. Hamsters were more sensitive to toxin A than mice or rats were. When hamsters were given multiple low doses of toxin A 1 week apart at a concentration which singly caused no response, they became ill and died, indicating that the toxin may have long-term effects. High amounts of toxin B did not cause any significant response when given intragastrically, unless initially mixed with low amounts of toxin A or given to hamsters with bruised ceca. These results suggest that toxins A and B act synergistically and that the action of toxin B may occur via the tissue damage caused by toxin A.