We evaluated a combined posttransplant strategy using antilymphocyte serum (ALS) at time of engraftment followed by low dose total lymphoid irradiation (TLI) and donor bone marrow cell (BMC) inoculation administered either intrathymically (IT) or intravenously (IV) in the vigorously rejecting strain combination DA into Lew recipients. Allograft survival was significantly prolonged with administration of ALS in combination with TLI and IT (105 +/- 28.6 days) or IV (106.8 +/- 28.6 days) BMC compared to administration of ALS combined with either TLI (17.8 +/- 0.4 days) or BMC (9.0 +/- 0.0 days), or TLI combined with BMC (1 1.5 +/- 0.5 days) (P < 0.000 1, experimental vs control animals). There was no difference in survival between those animals who underwent IT or IV BMC inoculation. Third-party (WF) BMC inoculation did not significantly prolong allograft survival (10.0 +/- 1.0 days). A mild to moderate cellular infiltrate was present in allograft tissue after 100 days. To further characterize these cells, cytokine mRNA expression in allograft tissue (> 100 days posttransplant) was evaluated using nonisotopic in situ hybridization. A similar cytokine profile was demonstrated in allograft tissue compared to naive and isograft tissue, except for a slight increase in IL-2 (P < 0.02, control vs IV BMC; P = NS, other groups). In summary, unresponsiveness was induced in a high-responder strain combination using a combined posttransplant strategy of ALS, TLI, and donor antigen either IT or IV. The cytokine profile of the graft infiltrating cells was similar to that of normal tissue. Unresponsiveness may be the result of functional inactivation of these cells.