The reactions of nitric oxide with hemoglobin play an important role in explaining the vascular biology of this free radical. It is perhaps surprising that the level of nitrosylhemoglobin (HbNO) in which NO is bound to the ferrous hemoglobin heme in whole human blood under basal and stimulated conditions is a matter of some controversy, with measurements ranging from <1 nM to close to 10 M. In order to examine HbNO levels in human blood by using EPR spectroscopy, we have developed a regression-based spectral analysis technique that has a detection level of about 200 nM HbNO. We have utilized this methodology to detect the level of HbNO under basal conditions and during NO inhalation. The major findings of this study are as follows. (i) HbNO can be accurately detected and quantified in whole blood with a detection limit of ϳ200 nM. The role of hemoglobin in the biochemistry, physiology, and pharmacology of nitric oxide (NO) 3 and nitrovasodilators is controversial (1-3). Understanding how hemoglobin can control NO-mediated responses is crucial in order to fully appreciate the physiological limitations of endothelial derived nitric oxide, the sequelae of nitric oxide delivery (either through nitric oxide donors, nitrovasodilators, or nitric oxide inhalation), and the vasoconstrictive propensity of hemoglobinbased blood substitutes. It is generally accepted that oxygenated ferrous hemoglobin (oxy-Hb) can convert nitric oxide to nitrate, through a reductive dioxygenation reaction, to form ferric hemoglobin (met-Hb) (4). This reaction is fast (k ϭ 5 ϫ 10 7 M Ϫ1 s Ϫ1 ) (5), irreversible, and generates an inert product. It is also universally accepted that nitric oxide can bind to unliganded ferrous hemoglobin (deoxy-Hb) to form a nitrosyl derivative (HbNO) at an almost equally rapid rate (k ϭ 2.6 ϫ 10 7 M Ϫ1 s Ϫ1 ) (6). Although HbNO is reasonably stable because of the slow ligand off rate, it can be plausibly argued that the NO has not been destroyed but has been preserved, if a viable mechanism for release and utilization can be established. Two major central areas of controversy are whether sufficient quantities of HbNO can be formed in vivo, and whether the formation of S-nitrosohemoglobin from HbNO represents a viable mechanism for NO reutilization (7-10). It has been argued that at oxygen concentrations at which hemoglobin is predominantly oxygenated, the small pool of hemes that are still in the high oxygen affinity state (R-state) but are unliganded will bind NO up to 100 times faster than has been appreciated previously. This would favor the formation of HbNO over met-Hb at oxygen saturation levels less than 99% (11, 12). This conclusion has been disputed in several laboratories, and methodological mixing problems with these experiments have been highlighted (13-16). Gladwin et al. (17) examined NO/hemoglobin reactions in vivo in humans by supplementing endogenous production with inhaled NO. These experiments indicated that the major hemoglobin-derived product formed during NO inhalation was met-Hb, together...