The goals of this in vitro study were to investigate effects of etomidate on endothelium-dependent relaxation induced by acetylcholine in rat aorta, and to elucidate the associated cellular mechanism. In endothelium-intact rings precontracted with phenylephrine 10−6 M, dose-response curves for acetylcholine (10−9 to 10−5 M) and calcium ionophore (10−9 to 10−6 M) were generated in the presence and absence of etomidate (5×10−6, 10−5 M). In endothelium-intact or -denuded rings precontracted with phenylephrine 10−6 M, sodium nitroprusside (10−9 to 10−6 M) dose-response curves were generated in the presence and absence of etomidate (10−5 M). Etomidate (5×10−6, 10−5 M) produced a significant rightward shift in the dose-response curves induced by acetylcholine (receptor-mediated endothelium-dependent agonist) and calcium ionophore A23187 (non receptor-mediated endothelium-dependent agonist). Etomidate (10−5 M) had no effect on sodium nitroprusside (endothelium-independent nitric oxide donor)-induced vasorelaxant response in both endothelium-intact and -denuded rings. These results indicate that etomidate at clinically relevant concentrations attenuates endothelium-dependent relaxation induced by acetylcholine by an acting at a site distal to the endothelial muscarinic receptor, but proximal to guanylate cyclase activation of vascular smooth muscle in rat aorta.