Background. Costimulation blockade has been shown to be effective in achieving donor-specific immune unresponsiveness in models of organ transplantation. This study represents the first application of blockade of the CD40 costimulatory pathway to a murine model of limb allotransplantation. Methods. Eighteen Balb/c mice (H-2Kd) were randomized to four groups. The control group (n=5) received syngeneic limb transplants from Balb/c donors. The experimental groups were recipients of limb allografts from C57Bl/6 mice (H-2Kb) and received either no treatment (n=5) or treatment with MR1 (hamster antimouse CD40 ligand monoclonal antibody) 500 μg intraperitoneally (IP) on days 0, 2, 4, 6, 14, 28, and 60 (n=5). A fourth group received myocutaneous allografts from C57Bl/6 donors and the same treatment with MR1 (n=5). Results. Untreated limb allografts were rejected at a mean of 9.6±1.1 days postoperatively. MR1-treated limb allografts underwent rejection of the skin component at a mean of 75±25 days whereas the musculoskeletal component survived to a mean of 222±84 days with two allografts surviving more than 10 months (P<0.001). The MR1-treated myocutaneous allografts were rejected after 16.2±2 days. All groups demonstrated acute rejection on histology except the treated limb allograft group, which was more suggestive of a chronic process. No chimerism was detected in this group by flow cytometry. Conclusions. CD40 costimulatory blockade significantly prolonged limb-allograft survival, and the bone-marrow component may have played an important role. Tolerance was not achieved, and histologic evaluation suggested chronic rejection as a possible cause of allograft loss.