The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood but activation of small- and intermittent-conductance Ca(2+)-activated K(+) channels (SKCa and IKCa) is important. Previous studies have suggested that the significance of IKCa depends on [Ca(2+)]out. Also it has been suggested that K(+) is important through localized [K(+)]out signaling causing activation of the Na(+),K(+)-ATPase and inward-rectifying K(+) channels (Kir). Here we tested the hypothesis that the modulating effect of [Ca(2+)]out on the EDH-like response depends on [K(+)]out. We addressed this possibility using isometric myography of rat mesenteric small arteries. When [K(+)]out was 4.2 mM, relaxation to acetylcholine (ACh) was stronger at 2.5 mM [Ca(2+)]out than at 1 mM [Ca(2+)]out. Inhibition of IKCa with TRAM34 suppressed the relaxations but did not change the relation between the relaxations at the low and high [Ca(2+)]out. This [Ca(2+)]out-dependence disappeared at 5.9 mM [K(+)]out and in the presence of ouabain or BaCl2. Our results suggest that IKCa are involved in the localized [K(+)]out signaling which acts through the Na(+),K(+)-ATPase and Kir channels and that the significance of this endothelium-dependent pathway is modulated by [Ca(2+)]out.