Allosteric regulation of nitrite reduction by deoxyhemoglobin is proposed to mediate nitric oxide (NO) formation during hypoxia. Nitrite is predominantly an anion at physiological pH raising questions about the mechanism by which it enters the red blood cell (RBC) and whether this is regulated and coupled to deoxyhemoglobin mediated reduction. We tested the hypothesis that nitrite transport by RBC is regulated by fractional saturation. Using human RBC, nitrite consumption was faster at lower fractional saturations consistent with faster reactions with deoxyheme. A membrane-based regulation was suggested by slower nitrite consumption with intact versus lysed RBC. Interestingly, upon nitrite addition, intracellular nitrite concentrations attained a steady state which despite increased rates of consumption did not change with decreasing oxygen tensions, suggesting a deoxygenation sensitive step that either increases nitrite import or decreases the rate of nitrite export. A role for anion exchanger-1 (AE1) in controlling nitrite export was suggested by increased intracellular nitrite concentrations in RBC treated with DIDS. Moreover, deoxygenation decreased steady state levels of intracellular nitrite in AE1-inhibited RBC. Based on these data we propose a model in which deoxyhemoglobin binding to AE1 inhibits nitrite export under low oxygen tensions allowing for the coupling between deoxygenation and nitrite reduction to nitric oxide along the arterial-to-venous gradient.