It is well documented that uridine 5′-triphosphate (UTP) is released from several cells surrounding vascular smooth muscle cells including endothelial cells, platelets and sympathetic nerve terminals, and generally considered that UTP regulates vascular tone through selective activation of G-protein coupled P2Y receptors (Burnstock G, 2006). However, here we present evidence supporting that UTP mediates contraction of vascular smooth muscle through P2X receptor activation. Namely, we found that extracellularly applied UTP (≥10μM) induced transient inward current in arterial(cerebral, mesenteric or aortic) myocytes, by use of whole-cell patch clamp configuration at a holding potential of -60mV. The current amplitude was enhanced dose-dependently, and current-voltage relationship of the current showed inward-rectification, which was similar to those evoked by activation of P2X channel. The current was also activated by high concentration of uridine 5′-diphosphate(UDP; 1mM), however, uridine 5′-monophosphate (UMP; 1mM) and uridine (1mM) were ineffective. Furthermore, the current induced by UTP (1mM) was inhibited by Gd3+ or La3+ (IC50=51.3μM or 21.8μM, respectively; n=3~5). Suramin and PPADS, both P2 receptor antagonists, also inhibited the current, and their IC50 were 0.43μM and 0.36μM, respectively (n=3~5). A previous study suggested that UTP activates TRPC3 channel through P2Y receptors in vascular smooth muscle cells(Reading SA et al., 2005). However, SKF96365(30μM) and 2-APB (100μM), which are potent TRPC3 antagonists, failed to inhibit the UTP-induced current (n=3~5). Application of α,β-methylene ATP (10μM), a potent P2X receptor agonist, slightly potentiated the UTP (1mM) -evoked current (13.1±5.4% (n=8) from the basal current), but inversely, application of UTP (1mM) did not facilitate the α,β-methylene ATP (10μM) -evoked current. Similar effect was observed in the tension recordings in endothelium-denuded rat aorta ring preparations. These results indicate that UTP activates the same receptor which is sensitive to α,β-methylene ATP. Moreover, intracellular application of GDPβS or GTPγS (each 1mM), which is G-protein inhibitor or activator, respectively, did not modify the current at all. This result also indicates that the current was independent of G-protein coupled receptors. Single channel analysis revealed that both UTP (1mM) and α,β-methylene ATP (10μM) activate channels with similar conductance (10.5pS) in the outside-out mode. RT-PCR and Western blot analysis showed high expression of P2X1 subtype in cerebral and mesenteric arteries and aorta. However, TRPC3 transcript was not expressed in mesenteric artery and aorta. In cerebral artery, a faint band was observed for TRPC3. Taken together, our results suggest that UTP regulates arterial tone through P2X receptor activation, but not through activation of P2Y receptor.