The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood but activation of small- and intermittent-conductance Ca2+-activated K+ channels (SKCa and IKCa) is important. Previous studies have suggested that the significance of IKCa depends on Ca2+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 Ca2+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 Ca2+out than at 1 mM Ca2+out. Inhibition of IKCa with TRAM34 suppressed the relaxations but did not change the relation between the relaxations at the low and high Ca2+out. This Ca2+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 Ca2+out.