The present study were designed to characterize the action mechanisms of acetylcholine (ACh)- induced endothelium-dependent relaxation in arteries precontracted with high K⁢ (70 mM). For this, we simultaneously measured both muscle tension and cytosolic free Ca2⁢ concentration ([Ca2⁢]i), using fura-2, in endothelium-intact, rabbit carotid arterial strips. In the artery with endothelium, high K⁢ increased both [Ca2⁢]i and muscle tension whereas ACh (10μM) significantly relaxed the muscle and increased [Ca2⁢]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM), ACh increased [Ca2⁢]i without relaxing the muscle. In the artery without endothelium, high K⁢ increased both [Ca2⁢]i and muscle tension although ACh was ineffective. 4-DAMP (10 nM) or atropine (0.1μM) abolished ACh-induced increase in [Ca2⁢]i and relaxation. The increase of [Ca2⁢]i and vasorelaxation by ACh was siginificantly reduced by either 3μM gadolinium, 10μM lanthanum, or by 10μM SKF 96365. These results suggest that in rabbit carotid artery, ACh-evoked relaxation of 70 mM K⁢-induced contractions appears to be mediated by the release of NO. ACh-evoked vasorelaxation is mediated via the M3 subtype, and activation of the M3 subtype is suggested to stimulate nonselective cation channels, leading to increase of [Ca2⁢]i in endothelial cells.