Background. Induction of antigen-specific unresponsiveness to grafts is the ultimate goal for organ transplantation. It has been shown that anergic T cells generated in vivo can be transferred as suppresser cells. Anergic cells generated in vitro have never been successfully used to prevent allograft rejection in vivo. We examined whether anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway can suppress allograft rejection in vivo. Methods. Anergic T cells were generated in vitro by the addition of anti-B7-1 and anti-B7-2 monoclonal antibodies (mAbs) to primary mixed lymphocyte reaction (MLR) consisting of C57BL/6 (B6) splenocytes as responder and irradiated BALB/c splenocytes as stimulator. We tested the ability of these cells to respond to various stimuli and to suppress alloreactive T-cell responses in vitro. For in vivo studies, 4×10⁷ anergic cells were injected intravenously immediately after transplantation of BALB/c islets under the renal subcapsular space of streptozotocin-induced diabetic and 2.5-Gy X-irradiated B6 mice. Results. Anergic cells treated with both mAbs in the primary MLR did not proliferate in secondary MLR against BALB/c and third-party C3H/He stimulators. The cells also failed to respond to immobilized anti-CD3 mAb, although they proliferated in response to concanavalin A or phorbol myristate acetate + ionomycin. The anergic state was reversed by the addition of exogenous IL-2. Furthermore, these cells suppressed the proliferation of naive B6 T cells against either the same (BALB/c) or third-party (C3H/He) stimulator cells. In in vivo studies, irradiated B6 mice rejected BALB/c islet allografts acutely with a mean survival time of 27.0±8.3 days , whereas two of six animals injected with the anergic cells accepted the allografts indefinitely (>100 days) with a mean survival time of 52.0±38.2 days. Conclusions. Anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway suppress islet allograft rejection after adoptive transfer. This procedure might be clinically useful for promoting allograft survival.