The phenotype and function of T lymphocyte cell lines established in vitro from kidney biopsies at the time of acute cellular rejection were studied using a nonhuman primate renal allograft model. Our objectives were to investigate the function and surface phenotype of cells that infiltrate renal allografts in animals that were untreated, that were given subtherapeutic cyclosporin, or that developed rejection after treatment with monoclonal antibodies to IL-2R B chain (CD25), immune cell adhesion molecule-1 (ICAM-1), or CD8. Lines from allograft biopsies and peripheral blood were expanded in vitro using solely human recombinant IL-2 and analyzed after 6-20 days in culture. We found that the large majority of cells cultured from cynomolgus allografts at the time of acute rejection or, when possible, assayed directly without culture, were CD3+4-8+ T lymphoblasts that possessed donor-specific cytolytic function and an NK-line, cytotoxic activity. In contrast, it was rarely possible to establish T cell lines exhibiting donor-specific cytotoxic activity from the blood except in the absence of immunosuppression or during CsA taper. A stable number of graft-derived CD4+8- cells was only observed in an unsuppressed animal 2 days after transplantation in the absence of manifest signs of rejection. Taken together, the above data indicate that similar T lymphocyte populations associated with allograft rejection are present in acutely rejecting allografts after the various types of immunosuppressive therapy. Since the infiltrating cells were similar to those obtained prior to therapy, recurrent rejection most likely represents cells that have escaped elimination. The T cells derived from monkey grafts differ from those from human renal allografts by the decreased frequency of CD4+ cells. Whether this difference is species-related or therapy-related is not known.