Abstract—We examined the hypothesis that ONOO−, a product of the interaction between superoxide (O2·−) and nitric oxide (NO), inhibits calcium-activated K+(KCa) channel activity in vascular smooth muscle cells (VSMCs) of human coronary arterioles (HCAs), thereby reducing hyperpolarization-mediated vasodilation. HCAs were dissected from right atrial appendages. The interaction of ONOO−with microvessels was determined by immunohistochemistry using a nitrotyrosine antibody. Strong staining was observed in arteries exposed to authentic ONOO−or to sodium nitroprusside (SNP)+xanthine (XA)+xanthine oxidase (XO). Dilation to 10−8mol/L bradykinin (BK) was abolished in vessels exposed to ONOO−(−2.5±8%;P<0.05) but not DC-ONOO−(65±8%). Reduced dilation to BK was also observed after application of XO and SNP. Dilation to NS1619 (KCachannel opener) was reduced in endothelial denuded arterioles treated with ONOO−. In isolated VSMCs, whole-cell peak K+current density was reduced by ONOO−(control 65±15 pA/pF; ONOO−42±9 pA/pF;P<0.05). Iberiotoxin had no further effect on whole-cell K+current. In inside-out patches, ONOO−but not DC-ONOO−decreased open state probability (NPo) of KCachannel by 50±12%. O2·−generated by XA+XO had no effect on BK-induced dilation and NPoof KCachannels. These results suggest that ONOO−, but not O2·−, inhibits KCachannel activity in VSMCs possibly by a direct effect. This mechanism may contribute to impaired EDHF-mediated dilation in conditions such as ischemia/reperfusion where increased activity of NO synthase occurs in the presence of excess of O2·−.