Regulatory cells may play a pivotal role in inducing and maintaining transplantation tolerance. We investigated the mechanism of anergic lymphocytes with regulatory cell potential generated in vitro by ICOS and CD 28 co-stimulatory blockades as a source of cellular therapy for treating allograft rejection. Anergic lymphocytes were generated by a mixed lymphocyte reaction consisting of DA splenocytes as the stimulator and Lewis splenocytes as the responder in the presence of anti-ICOS mAb and rCTLA-4I g. Immunoregulatory effects of these lymphocytes were evaluated by secondary MLR and using other various stimulations. DA heart was transplanted into 7.5 Gy-irradiated Lewis rat after intravenous administration of these cells and/or Lewis spleen lymphocytes. We observed that these lymphocytes were not only anergic to alloantigen and polyclonal stimulations but also exhibited regulative activity to inhibit the alloreactive T-cell response. Our adoptive transfer studies revealed that irradiated recipients that received both anergic lymphocytes and naIve Lewis lymphocytes had significantly prolonged DA cardiac graft survival (mean 17.5 days) compared with a group that received Lewis lymphocytes alone (mean 10.8 days). Furthermore, some of the recipients accepted the graft indefinitely after receiving anergic lymphocytes alone (>100 days). These results demonstrated that anergic lymphocytes with regulatory activities can be generated through blocking co-stimulatory signals, CD 28 and ICOS, simultaneously in vitro, and may advance a new immunomodulatory strategy for preventing allorejection in organ transplantation.