Donor-specific unresponsiveness to LEW heterotopic cardiac allografts was induced in WF rats following intrathymic inoculation of LEW splenocytes in conjunction with a single intraperitoneal dose of antilymphocyte serum. In contrast, LEW cardiac allografts were promptly rejected in WF recipients pretreated with an intravenous inoculation of donor splenocytes. Without transient immunosuppression with antilymphocyte serum neither intrathymic nor intravenous inoculation of splenocytes led to allograft survival. Substitution of antilymphocyte serum by a short course of cyclosporine did not permit allograft survival, suggesting that a T-cell-depleting regimen is crucial to tolerance induction by this protocol. The unresponsive state could be transferred to secondary syngeneic hosts by spleen cells from long-term recipients of intrathymic splenocytes and cardiac allografts but not by spleen cells from recipients of intrathymic splenocytes alone. This suggests that persistence of donor alloantigen from the graft is necessary for maintenance of the tolerant state. The unresponsive state after intrathymic inoculation of allogeneic splenocytes may be mediated through interaction of maturing host thymocytes with donor alloantigen.