Control of early acute xenograft rejection xenoreactions in the hamster-to-Lewis rat xenotransplantation model with cyclosporine (CsA) and leflunomide subdues early T-independent xenoreactivity and uncovers a late immune response that can be controlled by CsA alone. We had attributed this acquired responsiveness to CsA to the induction of tolerance of T-independent xeno-antibody responses in the recipient and recently reported that this tolerance is species-specific. Here we have further characterized the specificity and nature of this tolerant state. Lewis rats transplanted with either hearts, skin, kidney or spleen/pancreas from Golden Syrian hamsters were treated with leflunomide (5 mg/kg/day by gavage) for 14-21 days and CsA (20 mg/kg/day by gavage) continuously from the day of transplant. Some Lewis rats received a second graft of hearts or skin from Golden Syrian hamsters (day 21-30 after first transplant), and a third heart graft from Balb/c mice (day 60 after the first transplant). Serum was harvested and the titers of xenoreactive antibodies were quantified by flow cytometry. All grafts were harvested at the end of each experiment and examined by histological and immunohistochemical methods. The combination of CsA and leflunomide was able to completely inhibit the rejection of kidney, spleen and pancreas xenografts in this hamster-to-rat xenotransplantation model. In addition, only a transient treatment with leflunomide was necessary, and long-term graft survival could subsequently be maintained by CsA alone. Histological examination of these grafts at > 80 days post-transplantation indicated minimal signs of rejection. These immediately vascularized organs induced T-independent B-cell tolerance, so that second grafts of hamster hearts and skin could be maintained with CsA alone. Under the same immunosuppressive regimen, only four out of nine Lewis rats exhibited long-term hamster skin survival, probably reflecting the increased immunogenicity of the skin compared with other vascularized grafts. Nonetheless, all rats that did not reject the hamster skin graft also did not reject the hamster heart while on CsA alone. Finally, we demonstrate that the tolerant state could be maintained for up to 30 days in the absence of xenograft. The vigorous T-independent antibody response that mediates acute xenograft rejection in the hamster-to-rat model can be tolerized by the immunosuppressive regimen of CsA and leflunomide. The lack of organ specificity for the induction of this tolerance suggests that the xenoantigens inducing tolerance may be common endothelial cell antigens. Finally, the presence of the xenograft has been previously shown to be critical for the induction of T-independent B-cell tolerance, however, the tolerant state is relatively stable and persists after the removal of the xenograft.