Plasma hemoglobin (Hb) scavenges endothelium-derived nitric oxide (NO), producing systemic and pulmonary vasoconstriction in many species. We hypothesized that i.v. administration of murine cell-free Hb would produce pulmonary vasoconstriction and enhance hypoxic pulmonary vasoconstriction (HPV) in mice. To assess the impact of plasma Hb on basal pulmonary vascular tone in anesthetized mice we measured left lung pulmonary vascular resistance (LPVRI) before and after infusion of Hb at thoracotomy. To confirm the findings obtained at thoracotomy, measurements of right ventricular systolic pressure (RVSP) and systemic arterial pressure (SAP) were obtained in closed-chest wild-type mice. To elucidate whether pretreatment with Hb augments HPV we assessed the increase in LPVRI before and during regional lung hypoxia produced by left mainstem bronchial occlusion (LMBO) in wild-type mice pretreated with Hb. Infusion of Hb increased SAP but did not change pulmonary arterial pressure (PAP), left lung pulmonary arterial flow (QLPA) or LPVRI in either wild-type or diabetic mice with endothelial dysfunction. Scavenging of NO by plasma Hb did not alter HPV in wild-type mice. Inhibition of NO synthase with l-NAME did not change the basal LPVRI, but augmented HPV during LMBO. Our data suggest that scavenging of NO by plasma Hb does not alter pulmonary vascular tone in mice. Therefore, generation of NO in the pulmonary circulation is unlikely to be responsible for the low basal pulmonary vascular tone of mice.