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Fecal specimens from 223 subjects were evaluated for the presence of Clostridium difficile by use of a selective medium developed in our laboratory and for the presence of C. difficile cytotoxin. C. difficile and cytotoxin were detected in 89 and 83%, respectively, of patients with antimicrobial agent-associated pseudomembranous colitis (PMC). In patients in whom PMC was not documented, C. difficile and cytotoxin were present in only 37 and 21%, respectively. C. difficile and cytotoxin were also recovered from the feces of 6 and 3, respectively, of 13 antimicrobial recipients who did not have diarrhea. Although C. difficile appears to be a major cause of PMC, it is not responsible for at least some two-thirds of cases of antimicrobial agent-associated diarrhea in which PMC is not documented. Neither the recovery of C. difficile nor the detection of its cytotoxin should be considered diagnostic for C. difficile-induced disease.