Vaccines based on a highly conserved cell surface exposed C-repeat region of the group A streptococcal M protein molecule have been found to induce protection against mucosal challenge by homologous and heterologous streptococcal serotypes. Rabbit hyperimmune antisera were produced to four partially overlapping peptides of the C-repeat region of M6 protein. These were examined by both direct and competitive ELISA and by Western blotting for their reactivity against mammalian coiled coil proteins such as laminin, myosin, light meromyosin, heavy meromyosin, and cardiac tropomyosin, and to the denatured forms of some of these molecules. All sera reacted strongly with the recombinant M6 protein molecule. In addition, antibodies to three of the peptides displayed generally low levels of cross-reactivity with at least one of the mammalian proteins, whereas antibodies to one peptide did not cross-react with any of the proteins tested. The observed reactivity was found to be directed predominantly against denatured forms of the mammalian molecules. For instance, the cleaved forms of myosin bound better to the cross-reactive antibodies than the intact molecule. Furthermore, heat-denatured heavy meromyosin competed severalfold better in competitive ELISA than the non-heat-denatured "native" form. Our results demonstrate that M protein peptides corresponding to epitopes shared among rheumatic fever-associated strains of streptococci can lead to the production of low levels of antibodies reactive with mammalian coiled coil molecules. These antibodies are directed against the denatured forms of these molecules.