Nitric oxide (NO), synthesized by NO synthases (NOS), plays a pivotal role in regulation of pulmonary vascular tone. To examine the role of endothelial NOS (NOS3) in hypoxic pulmonary vasoconstriction (HPV), we measured left lung pulmonary vascular resistance (LPVR), intrapulmonary shunting, and arterial Po2 (PaO2) before and during left mainstem bronchus occlusion (LMBO) in mice with and without a deletion of the gene encoding NOS3. The increase of LPVR induced by LMBO was greater in NOS3-deficient mice than in wild-type mice (151 ± 39% vs. 109 ± 36%, mean ± SD; P < 0.05). NOS3-deficient mice had a lower intrapulmonary shunt fraction than wild-type mice (17.1 ± 3.6% vs. 21.7 ± 2.4%, P < 0.05) during LMBO. Both real-time PaO2 monitoring with an intra-arterial probe and arterial blood-gas analysis during LMBO showed higher PaO2 in NOS3-deficient mice than in wild-type mice ( P < 0.05). Inhibition of all three NOS isoforms with Nω-nitro-l-arginine methyl ester (l-NAME) augmented the increase of LPVR induced by LMBO in wild-type mice (183 ± 67% in l-NAME treated vs. 109 ± 36% in saline treated, P < 0.01) but not in NOS3-deficient mice. Similarly, systemic oxygenation during one-lung ventilation was augmented by l-NAME in wild-type mice but not in NOS3-deficient mice. These findings indicate that NO derived from NOS3 modulates HPV in vivo and that inhibition of NOS3 improves systemic oxygenation during acute unilateral lung hypoxia.