1. 1. In rat aortic rings contracted by phenylephrine, acetylcholine relaxation was partly inhibited by: iberiotoxin, a Ca 2+-activated K(K Ca) channel inhibitor; glyburide, an ATP-dependent K(K ATP) channel inhibitor; and 4-aminopyridine, a voltage-dependent K(K V) channel inhibitor, and was almost abolished by the removal of endothelium. 2. 2. N G-nitro- l-arginine (NOARG), a NO synthase inhibitor, markedly reduced acetylcholine relaxation and abolished the inhibitory effects of iberiotoxin and glyburide on the acetylcholine relaxation. The inhibitory effect of 4-aminopyridine on acetylcholine relaxation was partly reduced by NOARG. 3. 3. Methylene blue, a guanylate cyclase inhibitor, markedly inhibited acetylcholine relaxation and also abolished the inhibitory effects of iberiotoxin and glyburide and partly inhibited that of 4-aminopyridine on acetylcholine relaxation. 4. 4. Metyrapone, a cytochrome P-450-dependent monooxygenase inhibitor, and AA861, a 5-lipoxygenase inhibitor, but not indomethacin, a cyclooxygenase inhibitor, partly inhibited acetylcholine relaxation and reduced the inhibitory effect of 4-aminopyridine on acetylcholine relaxation. 5. 5. These results indicate that, in rat aortic rings, acetylcholine relaxation may be dependent on the activation of K Ca, K ATP and K V channels. The activations of K Ca and K ATP channels may also be dependent on NO synthesis and subsequent formation of cGMP. The activation of K V channels may also be dependent on NO synthesis and subsequent activation of guanylate cyclase. In addition, the activation of K V channels may be dependent on the metabolism of arachidonic acid through 5-lipoxygenase and cytochrome P-450-dependent monooxygenase pathways.