Descending vasa recta (DVR) are capillary sized microvessels that supply blood flow to the renal medulla. They are comprised of contractile pericytes and endothelial cells. In this study, we used the whole cell patch-clamp method to determine whether inward rectifier potassium channels (K_IR) exist in the endothelia, affect membrane potential and modulate intracellular Ca²⁺ concentration ([Ca²⁺]_CYT). The endothelium was accessed for electrophysiology by removing abluminal pericytes from collagenase-digested vessels. K_IR currents were recorded using symmetrical 140 mmol/L K⁺ solutions that served to maximize currents and eliminate cell-to-cell coupling by closing gap junctions. Large, inwardly rectifying currents were observed at membrane potentials below the equilibrium potential for K⁺ ion. Ba²⁺ potently inhibited those currents in a voltage dependent manner, with affinity, k = 0.18, 0.33, 0.60, 1.20 µmol/L at -160, -120, -80 and -40mV, respectively. Cs⁺ also blocked those currents with k = 20, 48, 253, 1856 µmol/L at -160, -120, -80 and -40mV, respectively. In the presence of 1 mmol/L ouabain, increasing extracellular K⁺ from 5 to 10 mmol/L hyperpolarized endothelial membrane potential by 15 mV and raised endothelial [Ca²⁺]_CYT. Both the K⁺ induced membrane hyperpolarization and the [Ca²⁺]_CYT elevation were reversed by Ba²⁺. Immunochemical staining verified that both pericytes and endothelial cells of DVR express K_IR2.1, K_IR2.2 and K_IR2.3 subunits. We conclude that strong, inwardly rectifying K_IR2.x isoforms are expressed in DVR and mediate K⁺ induced hyperpolarization of the endothelium.