A nonmyeloablative conditioning regimen has recently been developed that allows allogeneic marrow engraftment with induction of permanent mixed chimerism and donor-specific tolerance across fully MHC-mismatched allogeneic barriers. We recently demonstrated that tolerance can be broken in these chimeras by administration of an anti-donor class I-specific monoclonal antibody that eliminates donor hematopoietic cells. We have now investigated the role of the thymus in the loss of tolerance observed when chimerism is eliminated in this manner. Mixed chimeras were prepared in B10 (H2 b) recipients by treatment with depleting anti-CD4 and anti-CD8 mAbs, 3-Gy whole body irradiation, and 7-Gy thymic irradiation, followed by B10. A (H2 a) bone marrow transplantation. Chimeras were thymectomized 7 weeks later, and were either untreated or were depleted of donor cells with anti-donor class I (D d-specific) mAb 34-2-12. Control chimeras that were not thymectomized also received anti-donor monoclonal antibodies or no further treatment. Of the four groups, only euthymic animals that were depleted of donor antigen showed a loss of tolerance, as evidenced by rejection of B10. A skin grafts. In contrast to untreated control and thymectomized, anti-D d-treated chimeras, these euthymic anti-D d-treated chimeras showed significant recovery of Vβ11+ T cells, which can recognize Mtv antigens presented by donor IE molecules. The requirement for a thymus for loss of tolerance in the absence of donor antigen was verified in an adoptive transfer model, in which chimera (B10. A→ B10) spleen cells were depleted of donor-type cells ex vivo, adoptively transferred into B6 nu/nu mice, and then further depleted of donor-type antigen with monoclonal antibody treatment in vivo. These B6 nu/nu mice maintained donor-specific tolerance to B10. A skin grafts. The absence of active suppression as a potent mechanism of tolerance in long-term mixed chimeras was confirmed by the loss of mixed chimerism and of tolerance that was readily induced by injection of naive host-type spleen cells. Together, our results suggest that in mixed allogeneic chimeras, intrathymic clonal deletion, and not peripheral suppression or anergy, is the major mechanism maintaining donor-specific tolerance.