Extracellular nucleotides, such as ATP, UDP, and UTP, regulate pulmonary vascular tone through P2X and P2Y receptors. Recently, uridine adenosine tetraphosphate (Up4A) was reported as a novel endothelium-derived vasoconstrictive factor. Up4A contains both purine and pyrimidine moieties, which potentially activate P2X and P2Y receptors. The present study examined the effect of Up4A on contractility of isolated rat pulmonary artery. Up4A at 1–100 μM stimulated contraction in a concentration-dependent manner. Up4A was equipotent as UTP and UDP in the endothelium-denuded artery while much more effective than UTP and UDP in endothelium-intact preparations. The vasoconstrictor effect of Up4A was inhibited by suramin but not IP5I or desensitization of P2X receptors with α,β-methylene-ATP (α,β-Me-ATP). Up4A-induced contraction was also inhibited by pretreatment with thapsigargin, nitrendipine, or EGTA but unaffected by H1152. Furthermore, unlike ATP and UTP, Up4A did not induce relaxation of endothelium-intact preparations precontracted with phenylephrine. These results suggest that Up4A is a potent vasoconstrictor, but not a vasodilator, of the rat pulmonary artery. Up4A likely acts through a suramin-sensitive P2Y receptor. The contractile effect of Up4A involves the entry of extracellular Ca2+ and release of Ca2+ from intracellular stores but not Ca2+ sensitization via the RhoA/Rho kinase pathway. Up4A, therefore, potentially plays an important role in the regulation of pulmonary vascular tone.