Since intrathymic injection of UV-B-irradiated spleen cells induces donor-specific unresponsiveness in the sublethally irradiated (200 rads TBI) recipients, while intrathymic injection of naive SC leads to acute graft rejection, we hypothesized that presentation of MHC class I rather than MHC class II antigens to immature T cells in the thymus may convey a tolerogenic signal to the recipient. The present study was designed to examine if intrathymic injection of naive MHC class I-positive resting T lymphocytes can induce antigen-specific unresponsiveness to cardiac allografts in the Lewis-to-ACI rat combination. The results showed that intrathymic injection of resting Lewis T cells consistently induced indefinite graft survival (> 300 days) in sublethally irradiated (200 rads TBI) ACI recipients while similar treatment failed to prevent the rejection of third-party (Wistar-Furth) cardiac allografts, thus demonstrating the specificity of the immunologic unresponsiveness to donor alloantigens. Examination of the timing of intrathymic antigen presentation relative to cardiac transplantation that would achieve 100% permanent graft survival in the Lewis-to-ACI rat combination showed that the optimal time was 7 days before allografting, while peritransplant and immediate post-transplant intrathymic inoculation of donor T cells was relatively ineffective in the induction of unresponsiveness to donor grafts. We also showed that removal of the antigen-containing thymus in the sublethally irradiated recipients with functioning cardiac allografts consistently caused graft rejection if performed earlier than 21 days after heart transplantation; thymectomy after 21 days of organ transplantation did not affect indefinite survival of the grafts. Thus, it appears that the maintenance of peripheral tolerance to the grafts after 21 days of transplantation may be dependent on the presence of a new clone of antigen-specific tolerant host T cells. These results confirm the immunologic privileged position of the thymus in the induction of central and peripheral tolerance, and suggest that pretreatment with intrathymic MHC class I alloantigens is potentially useful in the induction of unresponsiveness to donor vascularized allografts in adult animals and in man.