In vitro susceptibility tests were performed on 84 strains of Clostridium difficile to 11 antimicrobial agents. All isolates were from the stools of patients with antibiotic-associated diarrhea or colitis in which there was a cytopathic toxin that was neutralized by Clostridium sordellii antitoxin. Over 95% of the strains were susceptible to vancomycin, penicillin G, ampicillin, and metronidazole at concentrations of 4 microgram/ml. Susceptibility to clindamycin was variable; 60% of the strains were susceptible at 1 microgram/ml, and 9% were resistant at 128 microgram/ml. Studies of individual isolates showed that a major portion of the strains were relatively susceptible to the antimicrobial agent implicated in causing the disease.