Prior studies from our laboratory have demonstrated that a nonmyeloablative conditioning regimen can induce transient mixed chimerism and renal allograft tolerance between MHC disparate cynomolgus monkeys. We have also shown that this preparative regimen can be extended to a concordant baboon to cynomolgus xenograft model by adding, to the post transplant protocol, therapy designed to prevent antibody production. Here we examine the use of brequinar (BQR) for this purpose and the efficacy of two new reagents developed to demonstrate the establishment of chimerism in the xenograft recipients. The cynomolgus recipients were conditioned with WBI (300 cGy), TI (700 cGy), ATG, cyclosporine, and brequinar sodium. To detect engraftment of the donor marrow, we prepared a polyclonal cynomolgus anti-baboon antibody (CABA) and a monoclonal antibody (215.1), which distinguish baboon and cynomolgus lymphocytes and granulocytes. We employed flow cytometry analysis to detect multilineage chimerism in the xenograft recipients. Five of the six recipients monitored using our new reagents (CABA and 215.1) developed detectable chimerism and only one of these animals lost its kidney to rejection. However, other complications have not permitted assessment of long-term outcome. The features of the multilineage chimerism included the detection of donor granulocytes (1.8-77.4%) and lymphocytes (2.4-22.2%) for 9 to 37 days. Our new reagents permit the detection of multilineage mixed chimerism, which may be a predictor of xenograft tolerance. We also conclude that brequinar may be effective in preventing antibody formation, but because of its toxicity, it is probably not the drug of choice for extension of the mixed chimerism protocol to concordant xenografts.