To examine the effect of chronic hypoxia on nitric oxide (NO) production and the amount of the endothelial isoform of nitric oxide synthase (eNOS) in lungs of newborn piglets, studies were performed using 1- to 3-day-old piglets raised in room air (control) or 10% O2 (chronic hypoxia) for 10-12 days. Exhaled NO output and plasma nitrites and nitrates (collectively termed NOx-) were measured in anesthetized animals. NOx- concentrations were measured in the perfusate of isolated lungs. eNOS amounts were assessed in whole lung homogenates. In the intact piglets, exhaled NO outputs and plasma NOx- were lower in the chronically hypoxic (exhaled NO output = 0.2 +/- 0.1 nmol/min; plasma NOx- = 10.3 +/- 3.7 nmol/ml) than in control animals (exhaled NO output = 0.8 +/- 0.2 nmol/min; plasma NOx- = 22.3 +/- 4.3 nmol/ml). In perfused lungs, the perfusate accumulation of NOx- was lower in chronic hypoxia (1.0 +/- 0.3 nmol/min) than in control (2.6 +/- 0.6 nmol/min) piglets. The amount of whole lung homogenate eNOS from the chronic hypoxia piglets was 40 +/- 8% less than that from the control piglets. The reduced NO production observed in anesthetized animals or perfused lungs of chronically hypoxic newborn piglets is consistent with the finding of reduced lung eNOS protein amounts. Decreased NO production might contribute to the development of chronic hypoxia-induced pulmonary hypertension in newborns.