Recipient pretreatment with UV-B irradiated donor-specific blood transfusions (UV-DST) combined with peritransplant cyclosporine on days 0, +1, and +2 leads to permanent cardiac allograft survival in the ACI-to-Lewis rat strain combination. This study investigates the mechanisms of immunologic unresponsiveness induced by UV-DST and CsA by examining several in vitro and in vivo parameters in long-term cardiac allograft recipients. The results of the in vitro studies demonstrate that thoracic duct lymphocytes (TDL) of treated and allografted Lewis rats respond less in a mixed lymphocyte reaction to donor splenic lymphocytes (SpL) by 69%, 75%, and 73% (P less than 0.001) at 30, 50, and 100 days after transplantation, respectively, compared with controls, while the response to a third-party (W/F) SpL is unimpaired. In coculture experiments, the TDL from treated recipients specifically suppressed the response of unmodified Lewis TDL to ACI SpL by 59% and 40% (P less than 0.01) at 30 and 50 days after transplantation, respectively, while responses to W/F SpL were suppressed by only 3-6%. The sera obtained from ungrafted rats transfused with UV-DST suppressed the MLR between unmodified Lewis TDL and ACI SpL by 31% (P less than 0.05) while the sera from UV-DST and CsA-treated and allografted rats specifically suppressed the MLR by 75%, 80% (P less than 0.001) and 37% (P less than 0.01) at 10, 30, and 50 days after transplantation, respectively. In vivo adoptive transfer of 10(4) donor-type dendritic cells (DC) into recipients of beating cardiac allografts at 40 or 60 days after transplantation led to rapid and acute allograft rejection, while the adoptive transfer of 10(8) unseparated SpL obtained at 50 days after transplantation from treated Lewis recipients to syngeneic naive hosts led to a modest but significant prolongation of ACI test cardiac allografts. The transfer of serum from treated and allografted recipients at 10, 30, and 50 days after transplantation led to specific and significant prolongation of test grafts in syngeneic naive hosts. These findings suggest that the mechanisms underlying the in vivo immunologic unresponsiveness induced by pretreatment with UV-DST and peritransplant CsA include inactivation without elimination of class II-antigen presenting cells (APC), generation of specific serum suppressor factor(s) and/or antiidiotypic antibody, and induction of donor-specific suppressor cells.