A model for induction of permanent PVG/c heart allograft survival in adult WKy rats, following antithymocyte globulin (ATG) treatment, was studied. Attempts to induce acute rejection of permanently accepted grafts, by injection of presensitized spleen cells, were successful only if the tolerant recipients had been pretreated with cyclophosphamide. Tolerance against second allografts from the primary donor strain could not be abrogated by injection of presensitized cells shortly after grafting. After removal of the permanently accepted graft, the tolerant recipients regained their capacity for acute rejection within 100 days.Tolerance was adoptively transferred with spleen cells, obtained from tolerant rats, to syngeneic sublethally irradiated recipients. The transferred unresponsiveness was donor-specific, antigen-dependent, and could again be transferred to irradiated recipients. The transferred suppression was abolished by the addition of presensitized, but not by naive, syngeneic spleen cells.The results indicate that donor-specific, antigen-dependent suppressor cells are essential for the maintenance of ATG-induced allograft acceptance. The suppression may influence the generation and activation, as well as the function, of alloreactive cells.