Removal of antimicrobial agents from formulary restriction status at our center was followed by an increase in the incidence of Clostridium difficile-associated diarrhea. The mean monthly incidence of C. difficile diarrhea for the 12-month period before institution of decreased restriction of antibiotic use was 3.4 infections per 1,000 admissions and rose to 6.2 infections per 1,000 admissions during the following 4-month period (P < .05). Patients who developed disease before and after decreased restriction of antibiotics were similar in terms of the mean number of antimicrobial agents administered and mean duration of therapy. The most commonly administered agents whose use preceded the onset of disease were cefazolin, trimethoprim-sulfamethoxazole, ampicillin, ticarcillin/clavulanate, and gentamicin (the latter drug was always used in combination with other agents). Immunoblot typing indicated that there was no association between C. difficile strains and administration of specific agents. There was no coincidental epidemic to account for the increased incidence of infection. The increased incidence of C. difficile disease is a potential problem that may occur following removal of extended-spectrum antimicrobial agents from formulary restriction status.