We explore the question of whether adenosine 5'-triphosphate (ATP) acts as an excitatory neurotransmitter in guinea-pig gastric smooth muscle. In an organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured in the presence of atropine and guanethidine. Under electrical field stimulation (EFS) at high frequencies (＞20 Hz), NO-mediated relaxation during EFS was followed by a strong contraction after the cessation of EFS (a "rebound-contraction"). Exogenous ATP mimicked the rebound-contraction. A known P2Y-purinoceptor antagonist, reactive blue 2 (RB-2), blocked the rebound-contraction while selective desensitization of P2X-purinoceptor with α,β-MeATP did not affect it. ATP and 2-MeSATP induced smooth muscle contraction, which was effectively blocked by RB-2 and suramin, a nonselective P2-purinoceptor antagonist. Particularly, in the presence of RB-2, exogenous ATP and 2-MeSATP inhibited spontaneous phasic contractions, suggesting the existence of different populations of purinoceptors. Both the rebound-contraction and the agonist-induced contraction were not inhibited by indomethacin. The rank orders of agonists' potency were 2-MeSATP ＞ ATP ≥ UTP for contraction and α,β-MeATP ≥ β,γ-MeATP for inhibition of the phasic contraction, that accord with the commonly accepted rank order of the classical P2Y-purinoceptor subtypes. Electrical activities of smooth muscles were only slightly influenced by ATP and 2-MeSATP, whereas α,β-MeATP attenuated slow waves with membrane hyperpolarization. From the above results, it is suggested that ATP acts as an excitatory neurotransmitter, which mediates the rebound-contraction via P2Y-purinoceptor in guinea-pig gastric antrum.