Human umbilical cord vessels (HUCV) release dopamine and nitric oxide (NO). This study aims to verify whether HUCV release nitrocatecholamines such as 6-nitrodopamine (6-ND). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to identify 6-ND release from HUCV rings incubated in Krebs-Henseileit's solution. Vascular reactivity of HUCV rings was tested (with and without endothelium integrity) by suspension of the rings in an organ bath under isometric tension and application of 6-ND and other known mediators. LC-MS/MS revealed a basal release of 6-ND from endothelium intact from both human umbilical artery (HUA) and vein (HUV). The endothelium intact release was inhibited by the pre-treatment with NO synthesis inhibitor L-NAME (100 μM). In contrast to dopamine, noradrenaline and adrenaline, 6-ND did not contract HUCV, even in presence of L-NAME or ODQ. 6-ND (10 μM) produced a rightward shift of the concentration-response curves to dopamine (pA2: 5.96 in HUA and 5.72 in HUV). Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND (10 μM). In U-46619 (10 nM) pre-contracted endothelium intact tissues, 6-ND and the dopamine D2-receptor antagonist haloperidol induced concentration-dependent relaxations of HUA and HUV. Incubation with the dopamine D1-receptor antagonist SCH-23390 (10 nM) abolished relaxation induced by fenoldopam but did not affect those induced by 6-ND. 6-ND is released by HUCV and acts as a selective dopamine D2-receptor antagonist in this tissue. This represents a novel mechanism by which NO may modulate vascular reactivity independently of cGMP production. •HUCV releases 6-ND but only in endothelium intact sections. The endothelium intact release was inhibited by the pre-treatment with L-NAME.•Unlike dopamine, noradrenaline and adrenaline, 6-ND did not contract HUCV, nor in the presence of L-NAME nor ODQ.•6-ND caused a rightward shift of the concentration-response curves to dopamine but did not affect the responses of noradrenaline and adrenaline.•6-ND caused a concentration-dependent inhibition of the contractions induced by the dopamine D2-receptor agonist sumanirole in HUCV.•6-ND is suggestibly acting as a dopaminergic antagonist, with selectivity for the D2-receptor.•6-ND represents a novel mechanism by which NO may modulate vascular reactivity independently of cGMP production.