Renal tubular epithelial cells can be induced to express potentially immunogenic levels of class II MHC antigens but fail to stimulate the activation of allospecific T lymphocytes. The current series of experiments was performed to determine whether the failure of lymphocyte activation in this system is caused by defective T cell costimulation. It was found that cultured renal epithelial cells expressed class II MHC antigens and the immunoregulatory adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and lymphocyte function-associated antigen-3 after stimulation with IFN-gamma, but that the B7 ligand for CD28 was not expressed. Mixed cell culture experiments were set up in which lymphocytes were mixed with IFN-gamma-treated allogeneic renal cells. Lymphoproliferation and IL-2 production were only observed if bivalent anti-CD28 antibodies were titrated into these cultures. The requirement for antigen stimulation was retained by these lymphocytes, as no proliferation was observed after stimulation by class II MHC antigen nonexpressing, resting renal cells. Further experiments demonstrated that the effectiveness of the anti-CD28 antibody-mediated signal was enhanced by cross-linking with a secondary anti-kappa-chain antibody. These data are consistent with the concept that a costimulatory signal generated by ligation of CD28 is of central importance to the development of an immune response to alloantigen. Furthermore, these results indicate that tubular epithelial cells within a rejecting renal allograft are unlikely to initiate direct activation of infiltrating allospecific lymphocytes.