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 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, plays an important role in the regulation of pulmonary vascular tone.