A sandwich preparation was obtained by placing a segment of the endothelium-free guinea pig coronary artery over the intact carotid artery with the objective being to determine whether the endothelium-dependent hyperpolarization by acetylcholine chloride (ACh) is mediated by a humoral factor. ACh hyperpolarized the sandwiched coronary smooth muscle membrane, the amplitude being larger than that of the carotid artery and smaller than that of the intact coronary artery. When spontaneously active smooth muscles (stomach antrum or portal vein) were used as the donor tissue, no electrical signal was transducted to the overlying coronary smooth muscles. In the sandwiched coronary artery, the ACh-induced hyperpolarization was not inhibited by ouabain, indomethacin, or nitroarginine. Pinacidil hyperpolarized the coronary smooth muscle membrane; the responses were inhibited by glybenclamide but not by tetraethylammonium chloride (TEA). The ACh-induced hyperpolarization was inhibited by TEA but not by glybenclamide. These results suggest that the ACh-induced hyperpolarization is mediated by an endothelium-derived humoral substance (endothelium-derived hyperpolarizing factor). Possible contribution of Ca-dependent K channels, but not ATP-sensitive K channels, to the endothelium-derived hyperpolarizing factor-induced hyperpolarization was considered.