Recent data show that intrathymic injection of allogeneic cells induces donor-specific unresponsiveness to allografts. There is also evidence to suggest that, in addition to recognizing intact MHC molecules, T cells can recognize processed MHC peptides, although the role of this indirect mode of allo-recognition in allograft rejection is unknown. We report that a single intrathymic injection of 100 micrograms of a mixture of eight 25-mer synthetic polymorphic class II MHC allopeptides, representing the full-length sequence of RT1.Bu beta and RT1.Du beta (WF) into incompatible (RT1l) LEW recipients, induced a state of long-term unresponsiveness to subsequent engraftment 2 days later of WF, but not third party (RT1n) BN renal allografts. Intrathymic injection of 100 micrograms of either RT1.Bu beta or RT1.Du beta peptide mixtures alone were insufficient to prolong renal allograft survival. Intravenous or intrasplenic injection of the allopeptide mixture did not affect renal allograft survival, establishing the role of thymic recognition of class II MHC allopeptides in inducing systemic unresponsiveness. The induction of intrathymic donor-specific unresponsiveness was abrogated if thymectomy was performed on the day of renal transplantation or 5 days later. PBLs from long-term surviving animals exhibited marked reduction of proliferation to WF, but not third party BN stimulator lymphocytes in the standard mixed lymphocyte response assay in vitro. These observations emphasize the role of recognition of processed MHC molecules in vascularized allograft rejection and confirm the role of the thymus in acquired systemic tolerance to alloantigens.