Immune interactions in the heart were studied using a murine model of myosin-induced autoimmune myocarditis. A T cell hybridoma specific for mouse cardiac myosin was generated from A/J mice and used to demonstrate that endogenous myosin/I-A k complexes are constitutively expressed on antigen-presenting cells in the heart. This T cell hybridoma, Seu.5, was used as a functional probe to identify a myocarditis-inducing epitope of cardiac myosin. Overlapping peptides based on the cardiac myosin heavy chain cx (myhcot) sequences were synthesized and tested for their ability to stimulate Seu.5 T cells. One peptide, myhcα(325-357) strongly stimulated the Seu.5 T cells, localizing the epitope to this region of the myhca molecule. Using truncated peptides, the epitope was further localized to residues 334-352. The myhccx(334-352) peptide strongly induced myocarditis when administered to A/J mice, which was histologically indistinguishable from that induced by myosin. The myhcα(334-352) epitope was present in cardiac myosin and not skeletal muscle myosins, providing a biochemical basis for the cardiac specificity of this autoimmune disease. Induction of myocarditis by this epitope was restricted to the myhcot isoform and not the myhc[3 isoform, suggesting there may be a difference in the efficiency of generating tolerance to these isoforms of cardiac myosin, which are differentially developmentally regulated. The myhcot(334-352) epitope bound to purified I-A k molecules in a similar manner to other I-Ak-restricted immunogenic epitopes, HEL(48-61) and R.Nase(43-56). Importantly, the myhcα(334-352) epitope was able to bind to I-A k molecules on the surface of antigen-presenting cells in a stable manner. These findings demonstrate that autoantigenic epitopes can behave in a dominant manner and constitutively bind to class II molecules in the target organ in a similar manner to foreign immunogenic epitopes.