Fifteen isolates of Clostridium difficile from hamsters and human patients were inhibited or killed by low concentrations of metronidazole, vancomycin, penicillin, and ampicillin; the isolates were often reesistant to tetracycline, cephalosporins, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, and aminoglycosides. Antibiotics to which C. difficile was susceptible were able to prevent or postpone the colitis caused by clindamycin in hamsters. Colitis could be produced by treatment of hamsters with any one of these antibiotics. Production of colitis not only involved selection of resistant variants, but in some instances seemed to result from the acquisition of organisms after treatment, their persistence despite treatment, or from subinhibitory cecal concentrations of antibiotic (explainable by either pharmacologic factors or enzymatic inactivation). As in humans, no organisms other than C. difficile have been implicated conclusively as etiologic agents of colitis in hamsters. Our results suggest it may be wise to use isolation precautions for patients with colitis caused by C. difficile.