Growing evidence suggests that nitrite, acting via reduction to nitric oxide by deoxyhemoglobin, may play an important role in local control of blood flow during hypoxia. To investigate the effect of hypoxia (arterial PO₂= 65 Torr) on the kinetic properties of nitrite a bolus injection of sodium nitrite (10 mg/kg) was given intravenously to normoxic or hypoxic newborn lambs and the time course of plasma nitrite ([NO₂^-]) and methemoglobin concentrations (MetHb%) measured. The in vivo kinetics of nitrite disappearance from plasma were biphasic and were not affected by hypoxia. Changes in methemoglobin, a product of the reaction between nitrite with hemoglobin, also did not differ with the level of oxygenation. Hypoxia potentiated the hypotensive effects of nitrite on both pulmonary and systemic arterial pressures. The disappearance of nitrite from plasma was equivalent to the increase in methemoglobin on a molar basis. In contrast, nitrite metabolism in sheep blood in vitro resulted in more than one methemoglobin per nitrite equivalent when blood oxyhemoglobin saturation was in a mid (HbO₂=50.3%) or high (HbO₂= 97.0%) oxygenated state. Under low (HbO₂ = 5.2%) oxygenation, significantly less than one mole of methemoglobin was produced per nitrite equivalent indicating that a significant portion of nitrite is metabolized through pathways not resulting in methemoglobin. These data provide support to the idea that the vasodilating effects of nitrite are potentiated under hypoxic conditions due to the reduction of nitrite to nitric oxide by deoxyhemoglobin.