An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to toxins A and B of Clostridium difficile was developed. Serum samples from 340 patients were tested for determination of the age-related prevalence of antitoxin. Antibody to toxin A was present in 64% of patients more than two years old and antibody to toxin B in 66% of patients more than six months old. A strongly positive ELISA value correlated with the presence of cytotoxicity-neutralizing antibody (P less than 0.001). Strongly positive ELISA values were obtained more commonly in convalescent sera from 16 patients with C difficile-induced colitis than in sera from the control population (antibody to toxin A, P less than 0.05; antibody to toxin B, P less than 0.001). Testing of paired sera revealed significant increases in the titer of IgG antibody to toxin A or B. Ten of the 16 patients with colitis had IgM titers of greater than or equal to 1:160 to one or both toxins. The data presented suggest that antibodies to toxins A and B are present in the majority of older children and adults and that patients with C difficile-induced disease develop serologic responses to one or both toxins.