Extracellular nucleotides uridine-5′-triphosphate (UTP) and adenosine-5′-triphosphate (ATP) induce contraction of gastric smooth muscle (SM). The dinucleotide uridine adenosine tetraphosphate (Up4A), an endothelium-derived contraction factor, induces vascular SM contraction. Its effect on gastric SM contractions, however, is unknown. We addressed the hypothesis that Up4A induces gastric SM contraction via a mechanism that may differ between circular and longitudinal muscle (CM and LM, respectively). CM and LM were isolated from rat gastric fundus for the measurement of isometric tension. Up4A induced transient contractile responses in both CM and LM, which were similar to those induced by ATP and UTP. Up4A failed to induce contraction of either LM or CM in the absence of extracellular Ca2+ or in the presence of nimodipine, an inhibitor of voltage-gated Ca2+ channels. P2X1, 2, 4, 5 and 7 and P2Y1, 2, 4 and 6 receptor expression was detected in gastric SM by reverse transcription-polymerase chain reaction. IP5I (a P2X receptor antagonist) and α,β-methylene-ATP (a P2X receptor agonist) had no effect on Up4A-induced contractions of either LM or CM, and α,β-methylene-ATP alone failed to induce a contractile response in either tissue. Suramin (a P2Y receptor antagonist), on the other hand, significantly inhibited Up4A-induced contraction of CM, but not LM. Up4A-induced contraction of CM, but not LM, was also inhibited by pretreatment with Y-27632, an inhibitor of Rho-associated kinase. We conclude that Up4A induces extracellular Ca2+-dependent contractions of rat gastric LM and CM, and Up4A-induced contraction of CM is mediated by suramin-sensitive P2Y receptors and subsequent activation of the Rho-associated kinase pathway. © 2013 IUBMB Life, 65(7):623–632, 2013.