Serum immunoglobulin G (IgG) class antibodies directed against toxins A and B of Clostridium difficile were studied using an enzyme-linked immunosorbent assay and a serum-neutralizing assay based on the MRC-5 tissue cytotoxicity assay. Of 185 individuals, 46 sera (24%) in the general population demonstrated IgG antibody, 36 (19.4%) against toxin A and 15 (8.1%) against toxin B. Antibody titer in the general population did not correlate with serum-neutralizing activity. Antibody prevalence fell with age (P = 0.58) over 50 years. Six of ten patients with acute primary episodes of C. difficile-associated diarrhea demonstrated antibody in convalescent-phase sera, predominantly directed against toxin B. Only two (28%) of seven patients with a history of relapsing C. difficile disease had demonstrable antibody.