Anti-myosin Abs are associated with inflammatory heart diseases such as rheumatic carditis and myocarditis. In this study, human cross-reactive anti-streptococcal/anti-myosin mAbs 1. C8, 1. H9, 5. G3, and 3. B6, produced from peripheral blood lymphocytes of patients with rheumatic carditis, and mAb 10.2. 5, produced from a tonsil, were characterized, and the nucleotide sequences of their V H and V L genes were analyzed. Human mAbs 1. C8, 1. H9, 10.2. 5, and 3. B6 reacted with human cardiac myosin while mAb 5. G3 did not. The mAbs were strongly reactive with N-acetyl-β-d-glucosamine, the dominant epitope of the group A streptococcal carbohydrate. mAb 1. H9 was moderately cytotoxic to rat heart cells in vitro in the presence of complement. The anti-myosin mAbs from rheumatic carditis were found to react with specific peptides from the light meromyosin region of the human cardiac myosin molecule. Anti-streptococcal/anti-myosin mAbs from normal individuals reacted with distinctly different light meromyosin peptides. The mAbs were encoded by VH3 gene segments V3-8, V3-23, and V3-30 and by the VH4 gene segment V4-59. The variable region genes encoding the anti-streptococcal/anti-myosin repertoire were heterogeneous and exhibited little evidence of Ag-driven somatic mutation.