AIMS: Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. METHODS AND RESULTS: TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4- carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation. CONCLUSION: The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.