Recent data have led us to hypothesize that selective activation of endothelial small- and/or intermediate-conductance, calcium-activated potassium channels (SK(Ca) and IK(Ca) channels, respectively) by the opener compounds NS309 and DCEBIO would augment stimulated nitric oxide (NO) synthesis and vasodilation in resistance arteries. Experimentally, ATP-evoked changes in membrane potential, cytosolic Ca(2+), and NO synthesis were recorded by patch clamp and microfluorimetry in single human endothelial cells. Agonist-evoked inhibition of myogenic tone in isolated, pressurized arterioles from rat cremaster skeletal muscle was analyzed by video microscopy. NS309 and DCEBIO enhanced ATP-evoked membrane hyperpolarization and cytosolic Ca(2+) transients, along with acute NO synthesis in isolated endothelial cells. The acetylcholine-mediated inhibition of myogenic tone (IC(50)=237 nM) was left-shifted in the presence of NS309 and DCEBIO (10, 100, and 1000 nM) to IC(50) values of 101, 78, and 43 nM; endothelial denudation inhibited this drug effect. L-NAME attenuated the acetylcholine-induced inhibition of myogenic tone but did not interfere with NS309 and DCEBIO-evoked vasodilation. Collectively, our data demonstrate that drug-induced enhancement of endothelial SK(Ca) and IK(Ca) channel activities represents a novel cellular mechanism to increase vasodilation of small-resistance arterioles, thereby highlighting these channels as potential therapeutic targets in cardiovascular disease states associated with compromised NO signaling.