1. Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. 2. Nucleotides evoked complete (adenosine 5′ triphosphate (ATP), uridine 5′ triphosphate (UTP), uridine 5′ diphosphate (UDP); >90%) or partial (adenosine 5′ diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. 3. The rank order of potency was: UDP∼UTP∼ADP>adenosine 5′-[γ-thio] triphosphate (ATPγS)>ATP, with respective pD(2) values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y(1) (ADP>ATP), P2Y(2) or P2Y(4) (ATP and UTP) and P2Y(6) (UDP) receptors. 4. P2Y(4) receptors were not involved, since P2Y(4)-deficient mice displayed unaltered responses to ATP and UTP. 5. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pK(b) for ATP (4.53±0.07) was compatible with literature, but the pK(b) for UTP (5.19±0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y(2) receptor subtype(s). 6. Further, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). 7. Finally, 2′-deoxy-N(6)-methyladenosine3′,5′-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. 8. Taken together, these results point to the presence of functional P2Y(1) (ADP), P2Y(2) (ATP, UTP) and P2Y(6) (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.