The induction of donor-specific tolerance would eliminate the risk of long-term immunosuppression while ensuring allograft function and survival. Male Buffalo (RT1b) rats were exposed to donor alloantigen by an intrathymic, intrasplenic, s.c., or i.v. injection of 25 x 10(6) syngeneic Buffalo (RT1b) or MHC fully mismatched Lewis (RT1l), ACI (RT1a), or UV-B irradiated Lewis (RT1l) splenocytes. The Buffalo recipients were given 1 cc of rabbit antirat antilymphocyte serum (ALS) i.p. at the time of the donor antigen injection, and 21 days later received a heterotopic Lewis or ACI heart transplant. Only intrathymic alloantigen injection induced a donor-specific tolerance which allowed the cardiac allograft to survive indefinitely (mean survival time [MST] > 176.8 days) in > 86% of the recipients without the need for further immunosuppression, whereas groups receiving antigen injections at other sites rejected cardiac allografts in control time (MST approximately 7.0 days). Histologic examination of long-term tolerated Lewis cardiac allografts revealed the presence of healthy cardiac myocytes without mononuclear infiltration. Buffalo rats with a long-term surviving Lewis cardiac allograft did not reject a second Lewis cardiac allograft (MST > 100.0 days), but rejected a heterotopic ACI cardiac allograft in normal time (MST approximately 7.0 days). By limiting dilution analysis (LDA), maturation of donor-specific CTLs (pCTL) from long-term recipient splenocytes was markedly diminished, whereas third party pCTL was not altered, and T helper-precursors were moderately decreased without alteration in the peripheral CD4+ and CD8+ phenotype frequencies. MLC responses of recipients with long-term surviving cardiac allografts to donor-specific and third party stimulation were not significantly different from naive controls. Microchimerism is unlikely because Lewis allograft survival was also prolonged (MST > 96.0 days) in rats receiving UV-B irradiated Lewis splenocytes which cannot proliferate. The absence of increased allograft survival after transfer of long-term recipient splenocytes into naive animals suggests that donor-specific suppressor cells are not present. Additionally, in vitro lymphocyte proliferative responses to mitogenic or allogeneic stimulation in MLC was not diminished by the addition of these long-term recipient splenocytes. This model emphasizes the importance of exposure of T cell precursors to foreign donor alloantigen in the thymic environment for the development of unresponsiveness to a donor-specific vascularized allograft.