["Acta Physiologica, Volume 231, Issue 4, April 2021. ", "\nAbstract\n\nAim\nEndothelium‐derived hyperpolarization (EDH)‐mediated response plays an essential role in the control of kidney preglomerular circulation, but the identity of the K+ channels involved in this response is still controversial. We hypothesized that large‐ (KCa1.1), intermediate‐ (KCa3.1) and small (KCa2.3) ‐conductance Ca2+‐activated K+ (KCa) channels are expressed in the endothelium of the preglomerular circulation and participate in the EDH‐mediated response.\n\n\nMethods\nWe study the functional expression of different K+ channels in non‐cultured, freshly isolated native endothelial cells (ECs) of rat intrarenal arteries using immunofluorescence and the patch‐clamp technique. We correlate this with vasorelaxant responses ex vivo using wire myography.\n\n\nResults\nImmunofluorescence revealed the expression of KCa1.1, KCa3.1 and KCa2.3 channels in ECs. Under voltage‐clamp conditions, acetylcholine induced a marked increase in the outward currents in these cells, sensitive to the blockade of KCa1.1, KCa3.1 and KCa2.3 channels respectively. Isometric myography experiments, under conditions of endothelial nitric oxide synthase and cyclooxygenase inhibition, showed that blockade either of KCa1.1 or KCa3.1 channels was able to reduce the endothelium‐derived vasorelaxation of isolated interlobar arteries, while their combined blockade completely abolished it. In contrast, blockade of KCa2.3 channels did not reduce this vasorelaxant response, despite being functionally expressed in the endothelial cells.\n\n\nConclusion\nThis study shows that KCa1.1 and KCa3.1 channels are functionally expressed at the renal vascular endothelium and play a central role in the EDH‐mediated relaxation of kidney preglomerular arteries, which is important in the control of renal blood flow and glomerular filtration rate.\n\n"]