We describe a new method to measure human serum antibody against streptococcal erythrogenic toxins that uses inhibition of lymphocyte mitogenicity of the toxins as the indicator. Sera from 53% of 53 Kawasaki disease patients contained specific inhibitory activity against A toxin, whereas only 15% had serum inhibitory activity against B toxin. The specific anti-A toxin serum inhibitor was found in 10% of 118 age-matched control patients suffering from various infections and allergic diseases (p = 0.001, compared to Kawasaki disease patients). Serum inhibitory activity was detected in a small number of patients with beta-hemolytic streptococcal infection (3/19) and in none of the age-matched healthy children (0/17). However, four of seven cord blood sera samples and five of 13 sera samples from healthy neonates contained the inhibitor, a result suggesting passive transfer from mothers. Most of the antimitogen-positive sera were also positive by ELISA of IgG antibody against A toxin, and IgG fractions of the positive sera remained positive in both assays. Thus, it is possible that the specific serum inhibitor detected by the antimitogen assay represents anti-A toxin antibody. The role of toxin-producing bacteria in the pathogenesis of Kawasaki disease remains to be investigated.