Mechanisms that regulate endothelial nitric oxide synthase (eNOS) expression in normal and hypoxic pulmonary circulation are poorly understood. Lung eNOS expression is increased after chronic hypoxic pulmonary hypertension in rats, but whether this increase is due to altered hemodynamics or to hypoxia is unknown. Therefore, to determine the effect of blood flow changes on eNOS expression in the normal pulmonary circulation, and to determine whether the increase in eNOS expression after chronic hypoxia is caused by hemodynamic changes or low oxygen tension, we compared eNOS expression in the left and right lungs of normoxic and chronically hypoxic rats with surgical stenosis of the left pulmonary artery (LPA). LPA stenosis in normoxic rats reduced blood flow to the left lung from 9.8+/-0.9 to 0.8+/-0.4 ml/100 mg/min (sham surgery controls vs. LPA stenosis, P < 0.05), but there was not a significant increase in right lung blood flow. When compared with the right lung, eNOS protein and mRNA content in the left lung was decreased by 32+/-7 and 54+/-13%, respectively (P < 0.05), and right lung eNOS protein content was unchanged. After 3 wk of hypoxia, LPA stenosis reduced blood flow to the left lung from 5.8+/-0.6 to 1.5+/-0.4 ml/100 mg/min, and increased blood flow to the right lung from 5.8+/-0.5 to 10.0+/-1.4 ml/ 100 mg/min (sham surgery controls vs. LPA stenosis, P < 0.05). Despite reduced flow and pressure to the left lung and increased flow and pressure to the right lung, left and right lung eNOS protein and mRNA contents were not different. There were also no differences in lung eNOS protein levels when compared with chronically hypoxic sham surgery controls (P > 0.05). We conclude that reduction of pulmonary blood flow decreases eNOS mRNA and protein expression in normoxic adult rat lungs, and that hypoxia increases eNOS expression independently of changes in hemodynamics. These findings demonstrate that hemodynamic forces maintain eNOS content in the normoxic pulmonary circulation of the adult rat, and suggest that chronic hypoxia increases eNOS expression independently of changes in hemodynamics.