Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release CO during incubation in Krebs' buffer. Acute hypoxia elicits isometric tension development (0.77±0.06 mN/mm) in pulmonary vascular rings treated with chromium mesoporphyrin (15 µmol/L; CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides, which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P<0.05) by endothelium removal, by CO (1-100 µmol/L) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L754,142 (10 µmol/L). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1 which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 µmol/kg, IV) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L745142 (15 mg/kg, IV). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.