alphaalpha-Cross-linked hemoglobin (alphaalphaHb) is an example of a hemoglobin-based oxygen carrier (HBOC) with significant cardiovascular activity. This may compromise the safety and efficacy of this HBOC by causing systemic hypertension and reducing blood flow to some organs. The present work is based on the hypothesis that incorporating antioxidant activity into an HBOC in the form of a covalently attached nitroxide may prevent these effects. We have tested this hypothesis by adding antioxidant activity to alphaalphaHb with 2,2,6,6-tetramethyl-piperidinyl-1-oxyl (Tempo) to create polynitroxylated alphaalphaHb (PN-alphaalphaHb). The new compound PN-alphaalphaHb acts as an antioxidant in our in vitro and in vivo assays. In this study urethane-anesthetized rats were hemorrhaged to a mean arterial pressure (MAP) of 35-40 mmHg and maintained for 30 min. Animals were resuscitated with solutions of (1) 10% PN-alphaalphaHb (43 mmHg), (2) 10% alphaalphaHb (43 mmHg), (3) 7.5% albumin (43 mmHg), (4) 300% Ringers lactate (RL), and (5) 0. 9% normal saline equal to the shed blood volume (SBV). Hemodynamics and regional blood circulation was measured at baseline, following hemorrhage, and at 30 and 60 min postresuscitation using a radioactive microsphere technique. Base deficit (BD) was measured at baseline, following hemorrhage, and at 60 min following resuscitative fluid infusion. Finally survival was determined as the time following resuscitation until secession of heart rhythm. Saline and 300% RL resuscitation did not improve BD, systemic hemodynamics, or regional blood circulation. PN-alphaalphaHb, alphaalphaHb, and albumin significantly improved these parameters, however, only PN-alphaalphaHb and alphaalphaHb improved survival. PN-alphaalphaHb was found to be less hypertensive than alphaalphaHb due to blunted increases in both cardiac output and systemic vascular resistance. This study demonstrates that, by using alphaalphaHb as a scaffold for polynitroxylation, improvement in vasoactivity and resuscitative efficacy may be possible. In conclusion, the addition of antioxidant activity in the form of polynitroxylation of a low molecular weight Hb (alphaalphaHb) may create a safe and efficacious resuscitative fluid.