Recent studies show that the adult immune system can be manipulated to accept allografts by the intrathymic (IT) inoculation of donor alloantigens (Ag). To make this model clinically applicable to cadaveric transplants, we have examined the effect of simultaneous or sequential IT injection of two disparate Ags on graft survival. In initial experiments, Wistar Furth (WF) rats received IT injections of a single or an admixture of 2 x 10(7) Lewis T cells and 2 x 10(7) BN T cells 7 days prior to cardiac transplantation. Untreated WF recipients acutely rejected single Lewis, BN, and ACI heart allografts, respectively, at times equivalent to rejection of double hearts while IT injection of single or double 2 x 10(7) Lewis and 2 x 10(7) BN T cells on day -7 also led to acute graft rejection. As expected, IT injection of donor-type resting T cells combined with 1 ml ALS recipient immunosuppression on day -7 led to donor-specific Lewis or BN permanent graft survival (> 200 days). The long-term unresponsive WF recipients challenged 100 days after cardiac transplantation with 2nd-set grafts specifically and permanently accepted 2nd-set donor cardiac allografts. IT injection of an admixture of 2 x 10(7) Lewis and 2 x 10(7) BN T-cells combined with 1 ml ALS on day -7 resulted in 100% permanent double (Lewis and BN) allograft survivals (> 150 days). Similar treatment led to acute rejection of third-party (ACI) grafts while the simultaneously transplanted Lewis hearts survived indefinitely (> 150 days). In the second group of experiments, unresponsive recipients of single (Lewis or BN) cardiac allografts were given IT inoculation of a second Ag in a sequential fashion 30 or 100 days after primary heart transplant. Unresponsive WF recipients of Lewis grafts accepted permanently (> 100 days) second BN grafts that were transplanted 30 or 100 days after primary Lewis graft and 7 days after IT injection of BN T-cells and 1 ml ALS. Similarly, unresponsive WF recipients of BN grafts accepted second Lewis grafts after IT injection of Lewis T cells combined with ALS, 30 or 100 days after primary BN grafts. To define the mechanism of peripheral tolerance in this model, we have shown that adoptive transfer of unseparated spleen cells from unresponsive recipients induces prolonged double (Lewis and BN) graft survivals (> 60 days) but not ACI grafts in sublethally irradiated (400 rads) syngeneic (WF) recipients, thus suggesting that maintenance of tolerance is, in part, dependent on suppressor/regulatory cell network.(ABSTRACT TRUNCATED AT 400 WORDS)