We compared the mechanisms of vasorelaxation of acetylcholine and of substance P with reference to K(+) channels, and analyzed pharmacologically the nature of endothelium-derived substance(s) other than NO and prostanoids in monkey and dog coronary arteries. Coronary arteries were isolated from monkeys and dogs, and the isometric tension of the artery strips was measured. In canine coronary artery strips treated with indomethacin plus N(G)-nitro- L-arginine ( L-NA) and partially contracted with prostaglandin F(2alpha), acetylcholine induced concentration-related relaxation, which was abolished by removal of the endothelium. The relaxation was markedly suppressed but not abolished in the strips exposed to high K(+) media. Charybdotoxin plus apamin potently inhibited the relaxation to the similar extent to that by high K(+) media, whereas glibenclamide or iberiotoxin had no effect. The relaxation was markedly inhibited by quinacrine, a phospholipase A(2) inhibitor, and ketoconazole, a selective cytochrome P450 (CYP) 3A inhibitor, but not by sulfaphenazole, a selective CYP 2C inhibitor. In contrast to acetylcholine, endothelium-dependent and indomethacin-plus- L-NA-resistant relaxation induced by substance P was not inhibited by high K(+) media, charybdotoxin plus apamin, or ketoconazole. Quinacrine and AA861, a 5-lipoxygenase inhibitor, inhibited the relaxation induced by substance P. In monkey coronary artery, acetylcholine-induced relaxation resistant to indomethacin plus L-NA was abolished by endothelial denudation and by treatment with high K(+) media, charybdotoxin plus apamin, progesterone and ketoconazole, but was not affected by iberiotoxin or sulfaphenazole. Substance P did not relax monkey coronary arteries. It is concluded that endothelium-dependent, nitric oxide- and prostanoid-independent relaxation induced by acetylcholine in monkey and dog coronary arteries are mediated by charybdotoxin plus apamin-sensitive but iberiotoxin-insensitive Ca(2+)-activated K(+) channel opening substance(s), which may be CYP3A-derived arachidonic acid metabolite(s). Contrasting to the response to acetylcholine, endothelium-dependent, indomethacin-plus- L-NA-resistant relaxation induced by substance P in dog coronary artery is not associated with K(+) channel opening, and may be mediated by 5-lipoxygenase product(s).