ABSTRACT The mechanism of hypoxia-induced activation of arterial KCa channel current and vasodilation is unknown. We investigated roles of the 20-hydroxyeicosatetraenoic acid (20-HETE) and generation of superoxide in hypoxia activation of KCa channel current in cerebral arterial muscle cells (CAMCs) and cerebral vasodilation. Patch clamp analysis identified a voltage- and Ca2+-dependent 238 -/+ 21 pS K+ current sensitive to inhibition by tetraethylammonium (TEA, 1 mM) or iberiotoxin (100 nM). Hypoxia (< 2% O2) reversibly enhanced the NPo of the 238 pS KCa current in cell-attached patches, but not in inside-out or outside-out membrane patches. Inhibition of CYP 4A activity increased the NPo of KCa single-channel current. Hypoxia reduced basal endogenous 20-HETE level and formation of 20-HETE in cerebral vessel homogenates as determined by LC/MS. The concentration of 20-HETE was reduced when incubated with the superoxide donor KO2. 20-HETE attenuated hypoxia-induced activation of the KCa current in CAMCs. Hypoxia did not augment the increase in NPo of KCa channel current induced by inhibition of CYP 4A activity. In pressure constricted cerebral arteries hypoxia induced dilation was attenuated by 20-HETE or by the KCa channel blocker TEA. Hypoxia caused generation of superoxide as determined by hydroethidine staining, fluorescent HPLC analysis and attenuation of hypoxia induced activation of KCa channel current by tempol These results suggest that hypoxia exposure of CAMCs results in generation of superoxide and reduction in endogenous level of 20-HETE in that may account for the hypoxia-induced activation KCa channel currents and cerebral vasodilation.