To identify tissue-cross-reactive antigens other than M protein in Streptococcus pyogenes, proteins of M-positive strains and an M-negative strain were probed in Western blots for reactivity with cross-reactive streptococcal monoclonal antibodies (MAbs) 36.2.2 and 54.2.8. A protein(s) near a molecular mass of 60 kDa in extracts of five group A streptococcal serotypes and the M-negative strain reacted with the MAbs. A study of human antibody responses to purified membranes of S. pyogenes indicated a hyperreactivity to a 60-kDa protein in acute rheumatic fever. Since MAbs 36.2.2 and 54.2.8 are known to cross-react with myosin or actin and streptococcal M protein, the data suggest that a homology or conformation is shared between the 60-kDa antigen and M protein. Therefore, the 60-kDa antigen is a new heart- or tissue-cross-reactive antigen of S. pyogenes that shares immunologic epitopes with but is distinct from M protein.