This study was carried out to determine the influence and site of action of N omega-nitro-L-arginine methylester, an L-arginine analogue, on basal pulmonary vascular tone and hypoxic vasoconstriction in neonatal pig lungs. We studied isolated lungs from pigs, age 14.5 +/- 0.5 (SD) d and weight 3.6 +/- 0.7 kg, perfused with autologous blood at a constant flow rate. The arterial-venous occlusion method was used to determine sites of action upstream and downstream of the double occlusion pressure (Pd) during baseline, infusion of acetylcholine, and ventilation of the lung with a hypoxic gas mixture. The measurements were then repeated during the three conditions described above after adding N omega-nitro-L-arginine methylester, a competitive inhibitor of nitric oxide synthase, to the blood. During control conditions, the vascular resistance was almost evenly divided upstream and downstream of Pd. Infusion of acetylcholine resulted in downstream dilation, and hypoxia resulted in an increase in both upstream and downstream resistance. After adding N omega-nitro-L-arginine methylester to the blood, there was an increase in both upstream and downstream resistances; acetylcholine infusion resulted in an increase in total vascular resistance, which was entirely due to upstream constriction; and the hypoxia response was much larger both upstream and downstream of Pd. These results suggest that nitric oxide synthase helped maintain a low level of basal pulmonary vascular tone both upstream and downstream of Pd in these neonatal pig lungs; that the vascular effect of acetylcholine was changed from downstream dilation to upstream constriction by N omega-nitro-L-arginine methylester; and that nitric oxide synthase activity modulated both the upstream and downstream vasomotor response to hypoxia.