The influence of pinacidil, an activator of ATP-sensitive K+ channels, on the membrane potential of endothelial cells from intact rat aorta and cultured endothelial cells was investigated. Pinacidil evoked a slowly developing sustained hyperpolariza-tion of endothelial cells from isolated artery with the amplitude of 15±4 mV from the resting membrane potential of –4Ш мВ. In contrast, in cultured endothelial cells pinacidil was without response. Diazoxide, another activator of ATP-sensitive K+ channels, in half of the cultured cells tested, evoked a slowly developing sustained hyperpolarization with the amplitude of 3 mV. The rest of the cells studied did not respond by membrane potential changes to diazoxide. It was suggested that high sen­sitivity of the membrane potential of in situ endothelial cells to potassium channels openers may represent a potent signaling mechanism influencing endothelial cell function upon stimula­tion of vascular KATP channels.