Background-A respiratory cycle for nitric oxide (NO) would involve the formation of vasoactive metabolites between NO and hemoglobin during pulmonary oxygenation. We investigated the role of these metabolites in hypoxic tissue in vitro and in vivo in healthy subjects and patients with congestive heart failure (CHF compared with relaxations induced at 95% (PϽ0.05), consistent with an allosteric mechanism of hypoxic vasodilation. We also measured transpulmonary gradients of NO metabolites in healthy control subjects and in patients with CHF. In CHF patients but not control subjects, levels of SNO-Hb increase from 0.00293Ϯ0.00089 to 0.00585Ϯ0.00137 mol NO/mol hemoglobin tetramer (Pϭ0.005), whereas HbFeNO decreases from 0.00361Ϯ0.00109 to 0.00081Ϯ0.00040 mol NO/mol hemoglobin tetramer (Pϭ0.03) as hemoglobin is oxygenated in the pulmonary circulation. These metabolite gradients correlated with the hemoglobin O 2 saturation gradient (PϽ0.05) and inversely with cardiac index (PϽ0.05) for both CHF patients and control subjects. Conclusions-We confirm that RBC-bound NO mediates hypoxic vasodilation in vitro. Transpulmonary gradients of hemoglobin-bound NO are evident in CHF patients and are inversely dependent on cardiac index. Hemoglobin may transport and release NO bioactivity to areas of tissue hypoxia or during increased peripheral oxygen extraction via an allosteric mechanism.