A functional requirement for all hemoglobin-based oxygen carriers (HBOCs) is the maintenance of the heme-iron in the reduced state. This is necessary for the reversible binding/release of molecular oxygen and minimization of methemoglobin (Fe+3) formation. Acellular hemoglobins are especially susceptible to oxidation and denaturation. In the absence of the intrinsic reducing systems of the red blood cell, the reduced heme-Fe+2 can be oxidized to form increasing levels of methemoglobin that can give rise to free radicals and oxidative cellular damage. If acellular HBOCs are to be utilized as red cell substitutes for oxygen delivery, these carriers must be stabilized in the plasma, the carrier medium. Normal plasma contains reducing components, such as ascorbic acid and glutathione, that can afford protection to these acellular HBOCs through electron-transfer mediated processes. For these components to provide effective reduction to an HBOC, a favorable reduction potential difference must exist between the reducing agent and the HBOC. Using a modified thin-layer spectroelectrochemical method, a determination of the formal reduction potential (vs. Ag/AgCl) of several oxygen carriers, including monomeric myoglobin, tetrameric HbA and HbS, chemically cross-linked HbXL99alpha, polymerized oxyglobin (FDA approved for canine anemia), and the natural cross-linked polymeric Lumbricus hemoglobin, have been determined. In contrast to the negative formal reduction potentials (-155 to -50 mV) obtained for Mb, HbA, HbS, HbXL99alpha, and oxyglobin, Lumbricus hemoglobin exhibited a positive formal reduction potential (approximately 100 mV). These results may help explain the greater effectiveness of the tested reducing agents to reduce met Lumbricus hemoglobin, compared to the other HBOCs, back to the required reduced form necessary for physiological binding/release of oxygen. Each reducing agent was capable of reducing met Lumbricus hemoglobin to the fully reduced state, although the kinetics of these reactions were different. HbA, HbXL99alpha, and oxyglobin were only partially reduced (10 to 37%) by glutathione, beta-NADH, and ascorbic acid under similar conditions.