Autoantibodies against myosin are associated with myocarditis and rheumatic heart disease. In this study, the antigenic cross-reactivity of myosin and N-acetyl-glucosamine (GlcNAc), the dominant epitope of Group A streptococcal polysaccharide, was examined. Six antimyosin monoclonal antibodies (MAbs) derived from mice with cardiac myosin-induced myocarditis were characterized. All MAbs cross-reacted with GlcNAc, mimicking a subset of MAbs derived from rheumatic carditis patients that bind both myosin and streptococcal polysaccharide. Variable (V) region gene usage was diverse, with five of six MAb heavy-chain V regions encoded by distinct members of the J558 family and the sixth encoded by a member of the VGAM3.8 family. Light-chain V-region segments were derived from the Vk1, Vk4/5, Vk10, and Vk21 families. These antimyosin, anti-GlcNac MAbs demonstrated several T-cell-dependent features: they were predominantly immunoglobulin G, were encoded by V-region genes expressed late in development, and displayed somatic mutation. A direct correlation between the extent of somatic mutation and the affinity for myosin was observed. Affinity for GlcNAc also increased with the frequency of mutation, demonstrating that affinity maturation can occur simultaneously for both self antigen and foreign antigen. Based on these observations, we immunized mice with GlcNAc coupled to bovine serum albumin and demonstrated that a T-cell-dependent response to GlcNAc leads to antimyosin reactivity. We speculate that the pathogenic antibody response in rheumatic carditis may reflect the conversion of a T-cell-independent response to GlcNAc to a T-cell-dependent cross-reactive response to GlcNAc and myosin.