Constitutive nitric oxide (NO) synthesis contributes to maintain low pulmonary vascular resistance in mice, but less is known about its effects on microvasculature at the alveolar level. Given its role in regulating microvascular responsiveness in various species, we determined the effects of acute NO synthesis inhibition on pulmonary arteriolar diameter and oxidative stress in anesthetized, open chest mice with ventilated lungs. NO synthesis was inhibited acutely by injecting L-NAME (0.1 mg/g body wt) into the pulmonary circulation via the right jugular vein. We examined subpleural microvessels (less than 50 μm in diameter) in the right lung, during brief periods when the lungs were statically inflated with O2. NADPH oxidase subunit, p47-phox translocation to the membrane, suggestive of NADPH oxidase activation, was assessed in lung lysates by immunoblot. L-NAME decreased arteriolar diameter by about 14% and increased the translocation of p47-phox compared to saline controls. Our data suggest that constitutive NO production decreases microvascular tone and inhibits NADPH oxidase in the ventilated mouse lung during physiological conditions. This study presents a reliable model for examining the role of NO synthesis in the responses of individual pulmonary microvessels in-vivo, and may provide valuable information on pulmonary microvascular responses to various pathophysiological circumstances.