Acetylcholine interacts with endothelial muscarinic receptors to enhance nitric oxide (NO) release and thereby cause vasodilation. The present study was designed to determine if this effect of acetylcholine is mediated by muscarinic M 3 receptors. Thoracic aortae were isolated from wild-type (WT) and M 3 receptor knock out (M 3R −/−) male mice, and endothelium-intact (I) and -denuded (D) aortic rings were bathed in physiological buffer. Preparations were utilized to examine the contractile response to phenylephrine (1×10 −8–3×10 −4 M added cumulatively) and the vasodilatory actions of acetylcholine (10 −8–10 −4 M), carbachol (10 −9–10 −4 M), ATP (3×10 −5 M) and the NO donor SIN-1 (10 −4 M), each added in the presence of phenylephrine. Endothelium-dependent vasodilatory effects of acetylcholine and carbachol were obvious in aortae isolated from WT mice (56.3±9.8% and 49.1±4.1% reductions, respectively, in phenylephrine-induced contraction; p<0.05), while acetylcholine and carbachol-associated relaxations observed in endothelium-intact M 3R −/− preparations (17.9±2.6% and 13.5±4.2% reductions, respectively) did not differ significantly from time-control values. ATP-induced, endothelium-dependent vasodilation was similar in preparations from M 3R −/− and WT mice, and SIN-1 elicited similar dilatory effects in intact and denuded WT and M 3R −/− segments. Phenylephrine concentration–response curves were shifted leftwards by removal of the endothelium in both groups (EC 50 values: WT-I/D—25.59±6.86/3.13±1.01×10 −7 M; M 3R −/−I/D—13.92±4.21/1.52±0.46×10 −7 M; both p<0.05); however, the phenylephrine response did not differ significantly when compared between the WT and M 3R −/− groups. These results indicate that the attenuated vasodilatory effect of acetylcholine in endothelium-intact aortae from M 3R −/− mice is due to the absence of muscarinic M 3 receptors, and thus suggest that in mouse aorta, muscarinic M 3 receptors play a major role in the endothelium-dependent acetylcholine-induced vasodilation.