Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood ex vivo. However, the sources of the nitrosylating species in vivo and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO2; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors ex vivo, while it was enhanced upon caveolin-1 deletion in vivo, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function.