6-Nitrodopamine (6-ND) is a novel catecholamine that is released from human umbilical cord vessels and Chelonoidis carbonaria aortic rings. The synthesis/release of 6-ND is inhibited by either pre-incubation of the vessels with the nitric oxide (NO) synthase inhibitor L-NAME or by mechanical removal of the endothelium. 6-ND causes powerful vasorelaxation, acting as a potent and selective dopamine D2-like receptor antagonist. Basal release of 6-ND from Panterophis guttatus endothelium intact and denuded aortic rings was quantified by LC-MS/MS. In order to evaluate the interaction of 6-ND with other catecholamines, aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. Endothelium intact aortic rings presented basal release of 6-ND, which was significantly reduced by previous incubation with L-NAME (100 μM). In endothelin-1 (3 nM) pre-contracted endothelium intact aortic rings, 6-ND (10pM-1 μM) and the dopamine D2-receptor antagonist L-761,626 (10 pM-1 μM) induced concentration-dependent relaxations, which were not affected by incubation with L-NAME but greatly reduced in endothelium-removed aortic rings. 6-ND (0.1-1 μM) produced significant rightward shifts of the concentration-response curves to dopamine in L-NAME pre-treated endothelium-intact (pA2 7.01) rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND (1 μM). The EFS-induced contractions of L-NAME pre-treated endothelium-intact aortic rings were significantly inhibited by incubation with 6-ND (1 μM). The results indicate that 6-ND released from Pantherophis guttatus aortic rings is coupled to NO release and represents a new mechanism by which NO can modulate vascular reactivity independently of cGMP production.