After previous observation of increased susceptibility to Clostridium difficile enterocolitis in hamsters fed an atherogenic, high-fat diet, a study was undertaken to examine experimental reproducibility of this disease. Hamsters were fed either the high-fat diet or a control diet, then orally challenged with a toxigenic strain of C. difficile. Hamsters fed the high-fat diet suffered 80% morbidity, which was statistically significant from the 11% morbidity of the control diet group (P < or = 0.05). The disease presented acutely, the most common presentation being sudden death. The most common lesions in the affected hamsters were necrohemorrhagic cecitis and cecal mucosal hyperplasia. Hepatic lipidosis was consistent in all hamsters fed the high-fat diet. Toxigenic C. difficile could be recovered from the cecum of most affected hamsters, and toxins A and B were detected in these ceca. Hamsters that were fed the high-fat diet and orally inoculated with a nontoxigenic strain of C. difficile before experimental challenge with a toxigenic strain were initially protected against disease. The protection decreased with each exposure to the toxigenic strain. Results of in vitro colonization-resistance studies indicated that the cecal flora from hamsters fed the high-fat diet and control diets inhibited C. difficile growth, suggesting that increased disease susceptibility was not the result of altered cecal flora.