Recent studies have shown that nitrite is an important storage form and source of NO in biological systems. Controversy remains, however, regarding whether NO formation from nitrite occurs primarily in tissues or in blood. Questions also remain regarding the mechanism, magnitude, and contributions of several alternative pathways of nitrite-dependent NO generation in biological systems. To characterize the mechanism and magnitude of NO generation from nitrite, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The addition of nitrite triggered a large amount of NO generation in tissues such as heart and liver, but only trace NO production in blood. Carbon monoxide increased NO release from blood, suggesting that hemoglobin acts to scavenge NO not to generate it. Administration of the xanthine oxidase (XO) inhibitor oxypurinol or aldehyde oxidase (AO) inhibitor raloxifene significantly decreased NO generation from nitrite in heart or liver. NO formation rates increased dramatically with decreasing pH or with decreased oxygen tension. Isolated enzyme studies further confirm that XO and AO, but not hemoglobin, are critical nitrite reductases. Overall, NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations.