Activation of endothelial cell (EC) small (S) and intermediate (I) conductance calcium-activated potassium channels (KCa) and current or molecular transfer via myoendothelial gap junctions underlies endothelium-derived hyperpolarization (EDH) leading to vasodilation. The mechanism underlying the KCa component of vasodilator activity and the characteristics of gap junctions are targets for the selective control of vascular function. In rat mesenteric artery, S/IKCa are critical for the hyperpolarization and repolarization phases of EDH, respectively, and are spatially associated with EC-EC and myoendothelial gap junctions, respectively. This differential functional S/IKCa activation and spatial localization suggests a causal relationship between channel activity and distribution; with such localizations representing a potentially selective target for control of vasodilator function and vascular tone. The present study addressed the question of whether the spatial separation of S/IKCa occurs in other selected model vascular beds commonly used to examine mechanisms of vascular function in rat and mouse. Tissues were obtained from anaesthetized (3g/kg urethane, i.p.) animals and SKCa and IKCa distribution examined using three different SK3 (SKCa) and three different IK1 (SK4; IKCa) antibodies, with conventional confocal immunohistochemistry. IK1 was diffusely expressed in the endothelium of rat and mouse cremaster and rat middle-cerebral artery. As in rat mesenteric artery, IK1 was also expressed in a punctate manner at myoendothelial gap junction sites in the mouse, but not in rat cremaster or middle-cerebral artery. SK3 was diffusely expressed in the endothelium of all three vessels and, as in the rat mesenteric artery, was also associated with adjacent EC-EC gap junctions in the rat and mouse cremaster. Marked heterogeneity exists within and between vascular EC S/IKCa expression, and may reflect different underlying functional mechanisms within and between these beds; thus representing potentially selective therapeutic targets for control of vascular tone.