Permanent donor-specific tolerance to tissue or organ allografts can be readily achieved without immunosuppression by administration of donor lymphohematopoietic cells to neonatal rodents. In adult recipients, however, induction of transplantation tolerance by this strategy generally requires intensive cytoablative conditioning of the recipient. We have now demonstrated that intrathymic inoculation of donor bone marrow or hepatic cells in conjunction with a single dose of antilymphocyte serum is effective in prolonging survival of DA rat orthotopic liver allografts in LEW strain recipients, which ordinarily rapidly reject such transplants. The unresponsive state achieved is donor-specific, as evidenced by the failure of intrathymic inocula of third-party WF cells to promote survival of LEW recipients of orthotopic DA liver allografts. Moreover, intravenous administration of the donor cells fails to extend liver allograft survival, demonstrating that the inoculum must be present in the thymus to promote unresponsiveness. Established DA liver allografts induced a state of systemic tolerance in LEW hosts, allowing their subsequent acceptance of donor-strain skin allografts. We hypothesize that the unresponsive state achieved by intrathymic inoculation of donor cells may result from the deletion or functional inactivation of alloreactive clones in a thymus bearing donor alloantigens. In this regard, cells of the macrophage/dendritic lineage (descendants of the bone marrow inoculum or hepatic Kupffer cells) may play a critical role by promoting thymic microchimerism and exerting modulatory effect on T cell development.