It is current understanding in the literature that acetylcholine (ACh) vasodilating effect is endothelium-dependent. Also, different endothelial substances such as NO, EDHF and prostanoids can mediate ACh-induced vasorelaxation in different vessels. Although ACh-induced relaxation of isolated rat aorta rings is mainly related with NO release it is also dependent on prostanoids because pretreatment with indomethacin shifted the ACh curve to the right, reducing maximal effect (Vizioli et al., J Smooth Muscle Res, 41: 271–281, 2005). Consequently, one should expect that nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibition should affect AChinduced vasodilation “in vivo”. To test this hypothesis we studied the effect of i.v. pretreatment with L-NAME (20 mg/kg), indomethacin (5 mg/kg) or its combination on the response to i.v. infusion of ACh in urethane-anesthetized rats. ACh infusion caused progressive blood pressure (BP) fall up to −40 mmHg, which was completely abolished by homatropine methylbromide (1 mg/kg). Although L-NAME significantly increased baseline BP (∼50 mmHg) indicating NOS blockade, the response to ACh was not significantly affected. Indomethacin shifted the ACh curve to the left, suggesting that COX blockade potentiates the response to ACh “in vivo”, as opposed to what was observed in isolated aortic rings. After the combined NOS and COX inhibition, the hypotensive response to ACh infusion was not significantly different from that observed prior to pretreatment. The present results disagree with those reported in isolated vessels and raise doubts on the mechanism actually involved in the hypotensive response to ACh “in vivo”.