We have recently described a non-myeloablative conditioning regimen permitting engraftment of allogeneic bone marrow in mice which involves administration of anti-CD4 (GK1.5) plus anti-CD8 (2.43) monoclonal antibodies in vivo, 3 Gy whole body irradiation, plus 7 Gy thymic irradiation. B10 (H-2b) mice prepared by this regimen and infused with unmanipulated B10.D2 (H-2d) bone marrow develop permanent mixed lymphohematopoietic chimerism and specific tolerance to donor skin grafts. We now demonstrate that mixed chimerism persists longer than 170 days in the lymphoid tissues including spleen, thymus and bone marrow of such animals, and that equivalent levels of donor chimerism are observed in both T and B cell compartments. In addition stable mixed chimeras were found to be unresponsive to host (B10) and donor (B10.D2) stimulator cells in mixed lymphocyte reaction and in cell mediated lympholysis assays, while responses to a third party (B10.BR, H-2k) were intact. Persistent chimerism was found to be necessary for the maintenance of skin graft tolerance in these animals, since in vivo depletion of donor cells by treatment with an anti-H-2d (34-2-12) monoclonal antibody resulted in the subsequent rejection of donor skin grafts. These studies demonstrate that mixed allogeneic chimeras produced using this regimen are specifically tolerant to donor in vitro and in vivo, and that persistence of donor chimerism is critical for the maintenance of tolerance.