Although ATP-MgCl2 produces beneficial effects following various adverse circulatory conditions, it remains unknown whether this agent restores the depressed endothelial cell function [i.e., the reduced release of endothelium-derived nitric oxide (EDNO) and endothelium-derived contracting factors (EDCF)] in a model of trauma-hemorrhage and resuscitation. To determine this, rats underwent laparotomy (i.e., trauma induced), were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of shed blood volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with four times the volume of maximal bleedout with RL, following which ATP-MgCl2 (50 μmol/kg body wt) or saline was administered. At 1.5 h postresuscitation, the aorta and superior mesenteric artery (SMA) were isolated, and dose-responses for acetylcholine (ACh, an endothelium-dependent vasodilator, via EDNO) and nitroglycerin (an endothelium-independent vasodilator) were determined. In addition, hypoxia- induced contraction, a process mediated by EDCF, was assessed. The results indicate that the decreased endothelium-dependent relaxation after hemorrhage (sham 94 ± 3 and 97 ± 3% vs. hemorrhage 64 ± 5 and 57 ± 11% at 10-5 M ACh in aorta and SMA, respectively, P < 0.05) was restored with ATP-MgCl2 treatment. In contrast, there was no significant difference in nitroglycerin- induced relaxation. Moreover, the decreased hypoxia-induced aortic contraction after hemorrhage (sham 221 ± 26 mg/ring vs. hemorrhage 124 ± 22 mg/ring, P < 0.05) was attenuated by administration of ATP-MgCl2. Because ATP-MgCl2 restores the depressed endothelial cell function, this agent appears to be a useful adjunct for protecting the endothelium after trauma and hemorrhage, even in the absence of blood resuscitation.