Diaspirin Cross-linked Hemoglobin (DCLHbTM), a hemoglobin-based oxygen carrier, improves regional blood circulation and systemic hemodynamics in normal and hemorrhaged rats. The action of DCLHb is partly mediated by its scavenging effect on nitric oxide. This study was undertaken to determine the effect of DCLHb on nitric oxide mechanism in hemorrhagic conditions. We studied the modulation of cardiovascular effects of DCLHb by a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) in hemorrhaged rats. The base deficit, survival time, oxygen consumption, and blood circulation to the brain, heart, gastrointestinal tract, and kidneys were determined in 1) DCLHb (100 mg/kg, intravenously (i.v.), 2) L-NAME (2 mg/kg, i.v.), 3) L-NAME (2 mg/kg, i.v.) + DCLHb (100 mg/kg, i.v.), and 4) L-arginine (100 mg/kg/h, i.v.) + DCLHb (100 mg/kg, i.v.) treated rats. Hemorrhage was induced in urethane-anesthetized male rats by bleeding them at a rate of approximately .5 to 1 mL/min, until a mean arterial pressure of 35–40 mmHg was achieved. This blood pressure was maintained for 30 min. Sham-operated nonhemorrhaged rats survived for > 300 min, whereas hemorrhaged rats survived for only 85 ± 31 min. Hemorrhage significantly increased base deficit and decreased oxygen consumption. A significant decrease in heart rate, mean arterial pressure, cardiac output, stroke volume, and in blood flow to the gastrointestinal tract and kidneys was observed after hemorrhage. Resuscitation with DCLHb produced a significant increase in survival time, oxygen consumption, heart rate, mean arterial pressure, cardiac output, total peripheral resistance, and blood flow to the brain, heart, and kidneys. In contrast, resuscitation with L-NAME did not improve base deficit, survival time, oxygen consumption, systemic hemodynamics, or regional blood flow. L-arginine pretreatment did not affect DCLHb-induced resuscitation of hemorrhaged rats. Furthermore, L-NAME (pretreated or co-administered) attenuated the resuscitative effect of DCLHb. These data suggest that nitric oxide mechanism may not be the only mechanism involved in the resuscitative effect of DCLHb.