The purpose of this study was to determine if a hemoglobin-based oxygen carrier, HBOC-201 (Hemopure, Biopure Corp), alters endothelial function and nitric oxide physiology when used for hemorrhagic shock. Female swine (Sus scrofa) underwent catheterization of the femoral, circumflex iliac, and pulmonary arteries. Control animals (n = 3) underwent instrumentation only. Study animals underwent hemorrhage to mean arterial pressure of 30 ± 5 mmHg, were maintained for 45 minutes, and resuscitated to the baseline mean arterial pressure for 4 hours. Resuscitation fluids included: shed blood (SB) (n = 8), lactated Ringers plus shed blood (LRSB) (n = 8), and HBOC (n = 8). At baseline, 1, and 4 hours after resuscitation, acetylcholine was infused into the proximal iliac artery and endothelial-dependent relaxation was measured with M-mode ultrasonography. Nitric oxide levels were determined using a chemiluminescent assay. HBOC, SB, and LRSB provided equivalent survival and resuscitation as measured by mean arterial pressures (65.3 ± 2.48 mmHg); pulmonary artery mean pressures (15.8 ± 0.84 mmHg); and lactate levels (1.22 ± 0.19 mmol/L). HBOC group animals required the lowest resuscitation volume (SB, 41.5 ± 3.5 mL/kg; LRSB, 76.4 ± 1.1 mL/kg, HBOC, 14.6 ± 2.1 mL/kg, p < 0.001). Response to acetylcholine was normal in the SB and LRSB groups, but the HBOC group had diminished acetylcholine response (29.5% endothelial-dependent relaxation end resuscitation, p < 0.001). Arterial nitric oxide levels did not differ between study groups (p = 0.69). HBOC might be an alternative resuscitation agent in patients with hemorrhagic shock. Resuscitation with HBOC requires less volume than blood or crystalloid. These data suggest HBOC-201 has a vasoconstrictive effect that cannot be attributed soley to nitric oxide scavenging. © 2004 by the American College of Surgeons.