Pre-graft priming of heart allograft recipients with donor strain blood induces tolerance in 100% of adult rats in the congenic LEW.1W to LEW.1A combination. This tolerant state is specific for donor MHC antigens as third-party blood transfusions fail to induce tolerance, and third-party skin grafts are promptly rejected by tolerant graft recipients. In this study we have characterized the immunodominant donor (RT1u) class I and II allogenic peptides which elicit an in vitro proliferative response to splenocytes from recipients (RT1a) undergoing acute rejection or tolerant to a LEW.1A cardiac allograft. Paradoxically, splenocytes from tolerant animals responded more vigorously to a broader set of donor peptides than splenocytes from rejecting animals. In addition, several of these peptides were observed to be stimulatory only for tolerant splenocytes. These findings suggest that regulatory cells may be involved in tolerance induction or maintenance and are selected by specific motifs, which could be utilized for manipulating the immune system of graft recipients.