It is well known that antibodies to N-acetyl-beta-D-glucosamine (GlcNAc) cross-react with cardiac valves, skin, and other host tissues. However, molecular targets of these antibodies have not been identified. For this reason, anti-streptococcal mAb cross-reactive with group A streptococci and heart proteins were studied for their reactivity with GlcNAc, the immunodominant epitope of group A streptococcal carbohydrate. Characterization of the mAb that recognized GlcNAc revealed that each mAb had its own unique antigen-binding profile and pattern of immunofluorescence on rat heart cells. In the ELISA and Western blot these mAb reacted with cytoskeletal and heart proteins such as actin, keratin, myosin, and vimentin, as well as with streptococcal recombinant M5 and M6 proteins. Binding of the mAb to cytoskeletal proteins was inhibited by GlcNAc conjugated with BSA in a dose-dependent manner, and the mAb preferentially reacted with high-density GlcNAc-BSA conjugates. Antigenic determinants on the proteins recognized by the mAb were resistant to sodium periodate and N-acetylglucosaminidase treatment, suggesting reactivity with peptide and not carbohydrate structures. On reaction of the mAb with a panel of synthetic streptococcal, viral, and myosin peptides, one of the mAb, 49.8.9, was found to react most strongly with a synthetic peptide sequence synthesized from the coxsackievirus B3 capsid protein VP1, which shows homology with and cross-reacts with sequences in the streptococcal M6 protein and human cardiac myosin. This most interesting mAb, previously shown to neutralize coxsackie viruses, recognized the amino acid sequence RRKLEFF, which may mimic the GlcNAc epitope. The data collected show that we have identified a new group of multireactive autoantibodies that recognize GlcNAc and cytoskeletal proteins, as well as defined peptide epitopes.