Wetested whether mitochondria function as the O2sensorunderlying hypoxic pulmonary vasoconstriction (HPV). In buffer-perfused ratlungs, rotenone, myxothiazol, and diphenyleneiodonium, which inhibitmitochondria in the proximal region of the electron transport chain (ETC),abolished HPV without attenuating the response to U46619. Cyanide andantimycin A inhibit electron transfer in the distal region of the ETC, butthey did not abolish HPV. Cultured pulmonary artery (PA) myocytes contract inresponse to hypoxia or to U46619. The hypoxic response was abolished while theresponse to U46619 was maintained in mutant(&rgr;0) PA myocytes lacking a mitochondrial ETC.To test whether reactive oxygen species (ROS) derived from mitochondria act assignaling agents in HPV, the antioxidants pyrrolidinedithiocarbamate andebselen and the Cu,Zn superoxide dismutase inhibitor diethyldithiocarbamatewere used. These abolished HPV without affecting contraction to U46619,suggesting that ROS act as second messengers. In cultured PA myocytes,oxidation of intracellular 2′,7′-dichlorofluorescin diacetate(DCFH) dye increased under 2% O2, indicating thatmyocytes increase their generation ofH2O2during hypoxia. This wasattenuated by myxothiazol, implicating mitochondria as the source of increasedROS during HPV. These results indicate that mitochondrial ATP is not requiredfor HPV, that mitochondria function as O2sensorsduring hypoxia, and that ROS generated in the proximal region of the ETC actas second messengers in theresponse.