Attenuation of endothelium-dependent relaxing factor (EDRF) release may contribute to adverse sequelae commonly seen after reperfusion of an acutely ischemic extremity. The purpose of this study was to identify the compound responsible for the EDRF activity in the extremity and to evaluate its modulation by ischemia and reperfusion. Isolated rat hindlimbs were perfused at constant pressure with an albumin-enriched crystalloid buffer. Increasing log dose infusions of acetylcholine and nitroprusside were used to measure endothelium-dependent (EDRF-mediated) and endothelium-independent vasoreactivity, respectively. Graded reductions in total vascular resistance were seen in response to both agonists in the control group (n = 11). In the postischemic group (n = 7), 60 minutes of normothermic ischemia and 10 minutes of reperfusion reduced endothelium-dependent vasodilation to acetylcholine by approximately 40% (p < 0.01). Endothelium-independent vasodilation to nitroprusside was unaffected. NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide release, attenuated vasodilation to acetylcholine (p < 0.01) but not nitroprusside in both control and postischemic groups. Endothelium-dependent vasodilation in the rat hindlimb, mediated by nitric oxide, was selectively impaired by injury from ischemia and reperfusion. Strategies designed to minimize postischemic attenuation of nitric oxide release may be beneficial in the management of acute limb ischemia.