Monoclonal antibodies that deplete cells carrying their target antigen are being used increasingly for immunosuppression in clinical and experimental transplantation. We have characterized a panel of rat antimouse CD4 monoclonal antibodies with the aim of establishing, in a vascularized organ transplant model, whether prolonged graft survival can be induced without recipient T cell depletion. The spatial relationship of the epitopes recognized by the anti-CD4 mabs was established. Mabs of the IgG2b isotype were found to profoundly deplete CD4+ T cells in vivo, whereas IgG2a mabs did not. The IgG2b anti-CD4 mab YTS191 and the IgG2a mab KT6 both blocked proliferation of C3H/He leukocytes in mixed leukocyte culture. Potent suppression of rejection and indefinite survival of cardiac allografts, mismatched for both major and multiple minor histocompatibility antigens (C57BL/10, H-2b into C3H/He, H-2k), was achieved with the IgG2b anti-CD4 mab YTS191 that depleted CD4+ T cells, (n = 9, median survival time (MST) greater than 100 days, P less than 0.001). The non-depleting IgG2a anti-CD4 mab, KT6, which had been shown to recognize and epitope on the CD4 molecule closely related to that recognized by YTS191 and to block comparably in MLC, was also shown to be capable of producing long-term cardiac graft survival in this strain combination (n = 6, MST greater than 100 days P less than 0.001). The kinetics of the KT6 therapy on the blocking of the CD4 molecule in vivo were investigated and shown to correlate with the effectiveness of the mab in prolonging graft survival.