BACKGROUND AND PURPOSE Studies in isolated preparations of vascular tissue (mainly resistance vessels) provide evidence that anandamide exerts vasorelaxation. The aim of the present work was to further characterize the mechanisms involved in the vascular response induced by anandamide in a conduit vessel, rat aorta. EXPERIMENTAL APPROACH Isometric tension changes in response to a cumulative concentration-response curve of anandamide (1 nM-100 micro M) were recorded in aortic rings from male Wistar rats. The involvement of a number of factors in this relaxation was investigated including endothelium-derived vasorelaxant products, cannabinoid and vanilloid receptors (transient potential vanilloid receptor-1 (TRPV1)), release of calcitonin gene-related peptide (CGRP), anandamide metabolism and the membrane transporter for anandamide. KEY RESULTS Anandamide caused a significant concentration-dependent vasorelaxation in rat aorta. This vasorelaxation was significantly inhibited by Pertussis toxin, by a non-CB1/non-CB2 cannabinoid receptor antagonist, by endothelial denudation, by inhibition of nitric oxide synthesis or inhibition of prostanoid synthesis via cyclooxygenase-2 (COX-2), by blockade of prostaglandin receptors EP4 and by a fatty acid amino hydrolase inhibitor. Antagonists for CB1, CB2, TRPV1 or CGRP receptors, an inhibitor of the release of endothelium-derived hyperpolarizing factor, and an inhibitor of anandamide transport did not modify the vascular response to anandamide. CONCLUSIONS AND IMPLICATIONS Our results demonstrate, for the first time, the involvement of the non-CB1/non-CB2 cannabinoid receptor and an anandamide-arachidonic acid-COX-2 derived metabolite (which acts on EP4 receptors) in the endothelial vasorelaxation caused by anandamide in rat aorta.