Nitrite reacts with deoxyhemoglobin to generate nitric oxide (NO). This reaction has been proposed to contribute to nitrite-dependent vasodilation in vivo and potentially regulate physiological hypoxic vasodilation. Paradoxically, while deoxyhemoglobin can generate NO via nitrite reduction, both oxyhemoglobin and deoxyhemoglobin potently scavenge NO. Furthermore, at the very low oxygen tensions required to deoxygenate cell-free hemoglobin solutions in aortic ring bioassays, surprisingly low doses of nitrite can be reduced to NO directly by the blood vessel, independent of the presence of hemoglobin; this makes assessments of the role of hemoglobin in the bioactivation of nitrite difficult to characterize in these systems. Therefore, to study the oxygen-dependence and ability of deoxhemoglobin to generate vasodilatory NO from nitrite we performed full factorial experiments of oxyhemoglobin, deoxyhemoglobin and nitrite and found a highly significant interaction between hemoglobin deoxygenation and nitrite-dependent vasodilation (P = <0.0002). Furthermore, we compared the effect of hemoglobin oxygenation on authentic NO-dependent vasodilation using a NONOate NO donor and found there was no such interaction, i.e. both oxy- and deoxyhemoglobin inhibited NO-mediated vasodilation. Finally, we show another NO scavenger, C-PTIO, inhibits nitrite dependent vasodilation under normoxia and hypoxia, illustrating the uniqueness of the interaction of nitrite with deoxyhemoglobin. While both oxyhemoglobin and deoxyhemoglobin potently inhibit NO, deoxyhemoglobin exhibits unique functional duality as an NO scavenger and nitrite-dependent NO generator suggesting a model, in which intravascular NO homeostasis is regulated by a balance between NO scavenging and NO generation that is dynamically regulated by hemoglobins oxygen fractional saturation and allosteric nitrite reductase activity