We have previously shown that myoendothelial gap junctions are more prevalent in distal than in proximal arteries of the rat mesentery. In the present study we have investigated the role of gap junctions in the mechanism of action of endothelium-derived hyperpolarizing factor (EDHF) in these same vessels following relaxation with acetylcholine. Arteries were pre-constricted with phenylephrine and concentration response curves to acetylcholine were constructed in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-5) M) and indomethacin (10(-5) M) to prevent effects due to the release of nitric oxide and prostacyclins. Nitric oxide was found to have only a small role in the relaxation of the proximal vessels and was not involved in the relaxations of the distal vessels. 18 alpha-Glycyrrhetinic acid (10(-5) M), a putative gap junction uncoupler, significantly reduced acetylcholine-induced relaxations by 50% in both proximal and distal vessels. Potassium channel antagonists, tetraethylammonium chloride (TEA; 10(-3) M) and barium chloride (10(-4) M), together abolished the dilatory response in the proximal mesenteric arteries, but did not completely block responses in the distal arteries. The data suggest that gap junctions contribute significantly to the acetylcholine-induced relaxation in both proximal and distal arteries of the rat mesentery. We hypothesize that the absence of a correlation between the role of gap junctions and the incidence of myoendothelial gap junctions in these same vessels is due to significant effects of the inhibitors on gap junctions located in the smooth muscle layers of the larger vessels.