Previously, we reported that essential oil of Croton nepetaefolius (EOCN) decreases blood pressure in normotensive rats, an effect that seems resulting from its vasodilatory action directly upon vascular smooth muscle. In the present study, we aimed to study the role of endothelium-nitric oxide pathway in the mediation of vasodilatory effects of EOCN and two of its constituents, methyleugenol and alpha-terpineol, using rat isolated thoracic aorta and mesenteric vascular bed preparations. EOCN (1-300 microg/mL), in a concentration-dependent manner, relaxed isolated endothelium-intact aortic rings precontracted with KCl 60 mM, with an IC(50) value of 26.7 (14.7-48.2) microg/mL. Either pretreatment of the tissue with L-NAME, a nitric oxide synthase inhibitor, or mechanical endothelium removal increased significantly the IC(50) value to 66.6 (52.7-84.1) or 105.6 (91.3-122.2) microg/mL, respectively. In endothelium-intact aortic rings precontracted with norepinephrine, EOCN (10-200 microg/mL) produced a vasorelaxant action which was decreased by the pretreatment of the aortic rings with methylene blue, a guanylate cyclase inhibitor. In mesenteric bed preparations perfused under constant pressure, EOCN reverted the reduction of mesenteric flow caused by KCl (60 mM), an effect that was attenuated by L-NAME. Vasodilator responses to EOCN in mesenteric bed preparations were mimicked by methyleugenol and alpha-terpineol, and were also significantly reduced in the presence of L-NAME. In conclusion, EOCN has vasorelaxant effects in both a resistance vascular bed and in a conduit artery. They seem attributed, at least in part, to the actions of its main constituents methyleugenol and alpha-terpineol and appear partially dependent upon the integrity of a functional vascular endothelium. Inhibition of other transduction pathways may be involved in the mediation of these effects.