We have recently demonstrated that mixed xenogeneic chimerism and donor-specific tolerance can be produced across a species barrier using a nonmyeloablative conditioning regimen (1). This regimen involves pretreatment of B10 mice with mAbs against CD4+, CD8+, Thy1+, and NK1+ cells, followed by a low dose (3 Gy) of whole-body irradiation and a higher dose (7 Gy) of local irradiation to the thymus and administration of T cell-depleted (TCD) F344 strain rat BMC. Although initial mixed chimerism and de novo maturation of donor rat T cells can be demonstrated in such animals, chimerism is gradually lost, and is no longer detectable by 6 months following BMT (1). When rat skin was grafted onto such animals 4 months following BMT, however, donor-specific skin graft survival was markedly prolonged, while non-donor type rat skin grafts were rapidly rejected (1). These results suggested that a state of donor-specific T cell tolerance existed, and that loss of chimerism was not due to a T cell-mediated immune mechanism. In order to evaluate the possibility that a humoral mechanism might mediate delayed loss of xenogeneic bone marrow grafts, we have now examined sera at various times for the presence of antibody against donor cells. Groups of animals not receiving the complete tolerizing mAb pretreatment regimen produced antidonor lymphocytotoxic antibody in response to BMT and skin grafting. Flow cytometric studies demonstrated high levels of IgM and of IgG of all subclasses against rat BMC and spleen cells in these control mice immunized by BMT. In contrast, such antibodies were not detectable in sera from animals receiving BMT following pretreatment with the tolerance-inducing mAb regimen. Furthermore, the tolerant animals did not develop cytotoxic antibodies or high levels of IgM or IgG against donor BMC after loss of hematopoietic chimerism. Donor-type skin grafts were eventually rejected, but rejection of these and repeat skin grafts did not lead to a cytotoxic antibody response. Low levels of rat BMC-binding IgM antibody were also detected in sera of tolerant mice, but the intensity of staining of rat BMC was lower than that of control animals receiving conditioning without BMT. These results suggest that a state of tolerance exists among cells responsible for T cell-dependent IgG antibody subclasses and natural IgM antibodies in animals receiving BMT following this nonmyeloablative conditioning regimen.