The pathogenic mechanisms for developing acute rheumatic fever after group A streptococcal pharyngitis are still poorly understood. The glycolytic enzyme enolase is one of the major proteins on the surface of group A streptococci. Herein, significant cross-reactivity was shown between streptococcal enolase and human enolase. Fluorocytometric analysis revealed that antistreptococcal enolase antibodies react with the enolase expressed on the surface of hematopoietic cells. Furthermore, the enolase on the leukocyte surface was found to be up-regulated by inflammatory stimuli. Evaluation of antibody titers indicated that serum samples from patients with acute rheumatic fever have higher levels of antibodies that react with the human and bacterial enolases than do serum samples from patients with streptococcal pharyngitis or healthy control subjects. These results show that streptococcal enolase is a novel cross-reactive antigen that may play an important role in the initiation of the autoimmune diseases related to streptococcal infection.