This study examined the role of intrathymic injection of allogeneic spleen cells in induction of donor-specific unresponsiveness to heart allografts in the Lewis-to-ACI rat combination. Intrathymic injection of naive Lewis SC led to rejection in naive or sublethally irradiated (200 rads TBI) ACI recipients at times equivalent to those obtained in control animals. Intrathymic injection of UV-B-irradiated Lewis SC, on the other hand, led to indefinite cardiac allograft survival (> 300 days) in sublethally irradiated ACI recipients; similar treatment failed to prevent rejection of third-party (Wistar Furth) cardiac allografts, which demonstrates the specificity of the immunologic unresponsiveness thus induced. The finding that intrathymic injection of untreated allogeneic SC does not prevent rejection of subsequently transplanted allograft suggests that modulation of major histocompatibility complex class II molecule by methods such as UVB may be critical to induction of unresponsiveness. Inoculation of UV-B donor SC in extrathymic sites (subcutaneous, intraperitoneal and intratesticular) did not significantly prolong graft survival in similarly prepared animals, thus confirming the privileged position of the thymus in the induction of tolerance. When the unresponsive recipients of cardiac allografts were made diabetic at 100 days and rechallenged with a second-set donor-type neovascularized pancreatic islet grafts, three of four animals accepted permanently (> 100 days) the islet grafts, thus indicating tolerance to donor alloantigens. To define the underlying mechanisms of specific tolerance in this model, in vitro MLR and in vivo adoptive transfer studies failed to demonstrate suppressor activity in the long-term cardiac allograft recipients. In contrast CML assays using 51Cr-release showed that T cells obtained from the unresponsive animals had no detectable cytotoxic activity to Con A-stimulated donor blast targets. The latter finding suggests clonal anergy or deletion of cytotoxic T cells to donor alloantigens. Our results confirm the role of the thymus as a privileged site for the induction and maintenance of specific immunologic unresponsiveness to organ allografts and suggest that this approach may be potentially useful in clinical transplantation of immediately vascularized allografts and neovascularized grafts.