Ag-specific activation of T lymphocytes requires two signals, one by the TCR and a second by costimulatory molecules. In a CD4+ T helper cell-dependent experimental autoimmune myocarditis model, we provide genetic evidence that cardiac myosin-induced autoimmune myocarditis and the production of IgG auto-Abs is dependent on functional T cells and did not occur in mice lacking the tyrosine kinase p56lck or the tyrosine phosphatase CD45. By contrast, animals lacking the T cell-costimulatory molecule CD28 (CD28 -/-) developed autoimmune heart disease, although at significantly lower severity than in heterozygous littermates, and produced IgG auto-Abs depending on the concentration of the autoantigen administered. In addition, the isotypes of IgG auto-Abs specific for cardiac myosin differed between CD28 +/- and CD28 -/- mice. Whereas CD28 +/- mice predominantly produced Th2-mediated IgG1 auto-Abs, CD28 -/- mice produced predominantly IgG2a. These data suggest that CD28 costimulation plays a crucial role in induction and maintenance of autoimmune heart disease and that CD28 expression is required for predominant Th2-IgG1 responses in an autoimmune setting.