Myocarditis is a common cause of dilated cardiomyopathy leading to heart failure. Chronic stages of myocarditis may be initiated by autoimmune responses to exposed cardiac Ags after myocyte damage. Cardiac myosin, a heart autoantigen, induced experimental autoimmune myocarditis (EAM) in susceptible animals. Although cardiac myosin-induced myocarditis has been reported in Lewis rats, the main pathogenic epitope has not been identified. Using overlapping synthetic peptides of the S2 region of human cardiac myosin, we identified an amino acid sequence, S2-16 (residues 1052-1076), that induced severe myocarditis in Lewis rats. The myocarditic epitope was localized to a truncated S2-16 peptide (residues 1052-1073), which contained a sequence identical in human and rat cardiac myosin. The S2-16 peptide was not myocarditic for three other strains of rats, in which the lack of myocarditis was accompanied by the absence of strong S2-16-specific lymphocyte responses in vitro. For Lewis rats, S2-16 was characterized as a cryptic epitope of cardiac myosin because it did not recall lymphocyte and Ab responses after immunization with cardiac myosin. Lymphocytes from S2-16 immunized rats recognized not only S2-16, but also peptides in the S2-28 region. Furthermore, peptide S2-28 was the dominant epitope recognized by T cells from cardiac myosin immunized rats. S2-16 was presented by Lewis rat MHC class II molecules, and myocarditis induction was associated with an up-regulation of inflammatory cytokine production. S2-16-induced EAM provides a defined animal model to investigate mechanisms of EAM and modulation of immune responses to prevent autoimmune myocarditis.