Almost all of the studies evaluating the effect of chronic hypoxia on lung nitric oxide production have been performed in adult animals. Because results of studies in adult lungs should not be extrapolated to represent the newborn lung, we performed studies to determine whether decreased nitric oxide production might be involved in the pathogenesis of chronic hypoxia-induced pulmonary hypertension in newborns. We kept newborn pigs in chambers filled with room air (control) or 11-12% O2 for either 3-5 (short) or 10-12 (long) days. Using isolated lungs, we measured pulmonary vascular responses to agents that either stimulate or inhibit the synthesis of nitric oxide. To define the vascular sites of altered production of nitric oxide, we applied the micropuncture technique and measured small venular pressures before and after treatment with a nitric oxide synthesis inhibitor. Pulmonary vascular responses to acetylcholine were blunted in chronically hypoxic piglets of both the short and long groups. The nitric oxide synthesis inhibitor had a different effect in the lungs of control piglets than in those of chronically hypoxic piglets of the long but not of the short group. For the long group, the nitric oxide synthesis inhibitors caused constriction of both arteries and veins in lungs of control but not of chronically hypoxic piglets. These findings support the idea that decreased pulmonary vascular nitric oxide production occurs with chronic hypoxia in newborn pigs and might therefore contribute to the pathogenesis of pulmonary hypertension in newborns.