Uridine adenosine tetraphosphate (Up 4A), a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorders and induces aortic contraction through activation of cyclooxygenases (COX). We and others demonstrated that activation of A 1 or A 3 adenosine receptors (AR) results in vascular contraction via thromboxane (TX) A 2 production. However, the mechanisms of Up 4A-induced vascular contraction in mouse aorta are not understood. We hypothesize that Up 4A-induced aortic contraction is through COX-derived TXA 2 production, which requires activation of A 1 and/or A 3AR. Concentration responses to Up 4A were conducted in isolated aorta. The TXB 2 production, a metabolite of TXA 2, was also measured. Up 4A (10 −9–10 −5 M) produced a concentration-dependent contraction >70%, which was markedly attenuated by COX and COX1 but not by COX2 inhibition. Notably, Up 4A-induced aortic contraction was blunted by both TX synthase inhibitor ozagrel and TXA 2 receptor (TP) antagonist SQ29548. Surprisingly, A 3AR deletion had no effect on Up 4A-induced contraction. Moreover, A 1AR deletion or antagonism as well as A 1/A 3AR deletion potentiated Up 4A-induced aortic contraction, suggesting a vasodilator influence of A 1AR. In contrast, non-selective purinergic P2 receptor antagonist PPADS significantly blunted Up 4A-induced aortic contraction to a similar extent as selective P2X 1R antagonist MRS2159, the latter of which was further reduced by addition of ozagrel. Endothelial denudation almost fully attenuated Up 4A-induced contraction. Furthermore, Up 4A (3 µM) increased TXB 2 formation, which was inhibited by either MRS2159 or ozagrel. In conclusion, Up 4A-induced aortic contraction depends on activation of TX synthase and TP, which partially requires the activation of P2X 1R but not A 1 or A 3 AR through an endothelium-dependent mechanism.