An NADPH-oxidase complex containing at least two protein components (gp91-phox and p22-phox) and a unique low redox potential (-245 mV) cytochrome b-245 is the source of superoxide generated for bacterial killing in neutrophils and has been suggested as the oxygen sensor in the carotid body. In pure cultures of smooth muscle cells from calf small pulmonary arteries (300 microns diameter) the presence of the 91 kD protein specific to this cytochrome was demonstrated by Western blot analysis with monoclonal antibody 48. Low-temperature-difference spectrophotometry of homogenates of these cells demonstrated the characteristic cytochrome b-245 spectrum when titrated between redox potentials of -152 and -345 mV, consistent with the low redox potential form. When these same cells were exposed to hypoxia (approximately 40 mmHg), superoxide production increased significantly from 1.4 +/- 0.2 to 73 +/- 12 nmoles.min-1 mg-1 protein. Hypoxic generation of superoxide was inhibited by the NADPH-oxidase inhibitor diphenyleneiodonium (DPI: 10 microM) but not by the mitochondrial inhibitor myxathiazole (10 microM). The hypoxic superoxide increase was significantly greater than that observed from smooth muscle cells from large pulmonary arteries or from large or small systemic arteries. Fluorescence immunocytochemistry revealed the presence of the NADPH-oxidase protein in the walls of pulmonary arteries in rat lung slices, and confocal microscopy showed the complex to be widely distributed in the vicinity of the arterial smooth muscle walls. In hypoxia or norepinephrine (NP)-induced vasoconstriction of pulmonary artery rings from cats, the sensitivity to inhibition by DPI was observed to be significantly greater for hypoxia (ED50 = 0.8 microM) than for NP-induced (ED50 = 13.4 microM) constriction. Together these observations demonstrate that the unique cytochrome b-245 containing NADPH-oxidase is present in pulmonary artery smooth muscle and that an NADPH-oxidase or NADH-oxidoreductase complex is activated to release superoxide by hypoxic conditions. It is concluded that a trans-membrane NADPH-oxidase is the most likely and that activation of this system may be involved in the initiation of hypoxic pulmonary vasoconstriction.