Background and purpose: Vascular endothelial and smooth muscle cell phenotypes may change dramatically after isolation and in cell cultures. This study was designed to investigate gap junctions coupling in an integrated intact preparation and to test if K-IR channels modulate resting membrane conductance in "in situ" endothelial cells (EC), and acetylcholine (ACh)-evoked relaxation of the rat superior mesenteric artery. Experimental approach: Whole cell blind patch recordings of ionic currents from in situ EC, dye-coupling experiments, and functional studies were performed in rat superior mesenteric artery. Key results: EC were dye-coupled through gap junctions. 18 beta-glycyrretinic acid (25 mu M) decreased outward and inward currents, the 80% decay of time and time constant of the capacitative transients, capacitance, and increased input resistance. Barium chloride (30 mu M) decreased resting and ACh-evoked inward currents, the sensitivity of ACh-evoked relaxation, and decreased both the sensitivity and the maximal relaxation to S-nitroso-N-acetyl penicillamine in arteries with, but not in arteries without endothelium. Conclusions: The present results suggest that the EC layer of this large artery is electrically coupled, and that K-IR channels regulate resting inward conductance, hence suggesting that they are of importance for resting membrane potential in in situ EC. Moreover, EC K-IR channels are involved in ACh-evoked relaxation. (C) 2011 Elsevier Inc. All rights reserved.