The interaction between inspiratory fraction of O2 (FIO2) and endogenous nitric oxide (NO) regulation of pulmonary vascular tone was examined in intact anesthetized dogs. Stimulus (FIO2 of 1, 0.4, 0.21, 0.12, and 0.1)-response (changes in pulmonary artery pressure minus pulmonary artery occlusion pressure) curves were constructed with cardiac output kept constant (by opening a femoral arteriovenous bypass or inflating an inferior vena cava balloon catheter), before and after administration of compounds acting at different levels of the L-arginine-NO pathway, NG-nitro-L-arginine (L-NNA, 10 mg/kg iv, n = 16), a NO synthase inhibitor, and methylene blue (8 mg/kg iv, n = 16), a guanylate cyclase inhibitor. L-NNA and methylene blue did not influence pulmonary vascular tone in hyperoxic and in normoxic conditions, but they increased it during hypoxia, thus enhancing the vasopressor response to hypoxia (from 4.5 +/- 0.9 to 10.4 +/- 1.2 mmHg and from 4.2 +/- 0.8 to 9 +/- 1.5 mmHg, respectively, both P < 0.01). Hypoxic pulmonary vasoconstriction was augmented in dogs with a baseline hypoxic response (“responders”) and restored in dogs without hypoxic response (“nonresponders”). These results suggest that endogenous NO does not influence hyperoxic and normoxic pulmonary vascular tone, but that it inhibits hypoxic pulmonary vasoconstriction in intact anesthetized dogs.