Several mouse strains are susceptible to immunopathic myocarditis after infection with Coxsackievirus B3 (CB3). This disease is associated with autoantibodies that are directed against myosin. In this study we characterized sera from CB3-infected mice for their reactivity with three different myosin isoforms (heart, skeletal muscle, and brain myosins) and for autoantibody isotype by using an ELISA. Competitive inhibition assays and absorption studies with various myosins demonstrated the presence of two autoantibody populations in sera of susceptible A.CA and A.SW mice. The first was specific for cardiac myosin and was mainly IgG. The second antibody population cross-reacted with heart, skeletal muscle, and brain myosin and was mainly IgM. B10.PL/SgSf and B10.A/SgSf mice, which do not develop immunopathic myocarditis, produced only the IgM autoantibody population cross-reactive with all three myosin isoforms. Because the heart-specific myosin autoantibodies were found exclusively in the mouse strains that developed immunopathic myocarditis, they can be considered a serologic marker for autoimmune heart disease.