Functionally intact coronary artery segments studied in vitro responded to 15 min of hypoxia with relaxations of preexisting contractions. The hypoxic relaxations were obtained in preparations routinely denuded of endothelium and were unaffected by tetrodotoxin, by indomethacin or by the blockers of calcium-dependent potassium channels, apamin and charybdotoxin. Relaxations from contractions to the calcium channel opener Bay K 8644 and to spontaneous tone were attenuated most by hypoxia, and those to carbamylcholine and 5-hydroxytryptamine were inhibited to an intermediate extent. Contractions to the thromboxane A2 analog U 46619, dependent largely on intracellular calcium, were the least reduced during 15 min of hypoxia. Pretreatment of contracted preparations with glibenclamide, the potent antagonist of ATP-dependent potassium channels, before exposure to 95% N2/5% CO2 significantly attenuated, but did not eliminate, hypoxic relaxations. Hypoxic relaxations from contractions to the calcium channel opener Bay K 8644 and to spontaneous tone were antagonized most by glibenclamide, and those to U 46619 were reduced the least. In the presence of the calcium channel antagonist nifedipine, tissues contracted with carbamylcholine or 5-hydroxytryptamine relaxed during hypoxia, but these relaxations were insensitive to glibenclamide. Contractions of cattle radial artery and rabbit aorta were variably reduced during hypoxia but were insensitive to glibenclamide. We conclude that K+ ATP channels participate in hypoxia-induced coronary artery smooth muscle relaxation and may do so particularly with contractions that utilize principally extracellular calcium.