Posttransplant infusion of viable donor bone marrow cells (DBMC) has been shown in our previous studies to promote acceptance of incompatible kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes. In this nonhuman primate model, the infusion of the DBMC is requisite for the induction of functional graft tolerance and specific MLR and CTLp unresponsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain. Standard immunological and molecular techniques applied to this monkey model are unable to differentiate between chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants. To distinguish chimerism due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neo(r)) using the retroviral vector pHSG-Neo.Neo(r)-transduced BMC were infused into recipients approximately 2 wk after kidney transplantation and treatment with rabbit antithymocyte globulin. No maintenance immunosuppressive drugs were given. Genomic DNA isolated from peripheral blood leukocytes was used to monitor the presence ofNeo(r)-positive cells. Tissue samples obtained at necropsy also were assessed forNeo(r)-positive chimeric cells. The presence of DBMC-derived chimerism was assessed by polymerase chain reaction usingNeo(r) sequence-specific primers (PCR-SSP). Chimerism was detectable in recipient tissues at various times for up to 6 mo after DBMC infusion. These studies using gene transduction methodology indicate that a stable genetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a nonhuman primate model. This approach should facilitate future studies in preclinical models to study the role and type of chimeric cell lineages in relation to functional allograft tolerance.