We investigated the role of the 5-hydroxytryptamine3 (5-HT3) receptor in the regulation of gastric emptying in rats using various 5-HT3 receptor antagonists, including GK128, a novel and selective 5-HT3 receptor antagonist. GK128 dose-dependently accelerated gastric emptying in rats. The accelerating effect of GK128 on gastric emptying was more potent than that of the other 5-HT3 receptor antagonists used in this study. However, the rank order of potency of the selective 5-HT3 receptor antagonists, except for the benzamide derivatives, on the accelerating effect of gastric emptying, was not consistent with that of their 5-HT3 receptor-binding affinity in the rat cortex. GK128 improved the gastric emptying delayed by m-chlorophenylbiguanide, a 5-HT3 receptor agonist, and by cisplatin, which is known to cause damage to the small intestine and to release 5-HT from enterochromaffin cells. Furthermore, 5,7-dihydroxytryptamine, an indoleamine neurotoxin known to destroy 5-HT-containing neurons, significantly accelerated gastric emptying, and no further acceleration was observed after administration of GK128. These results may suggest that 5-HT3 receptor antagonists induce, at least in part, the acceleration of gastric emptying in rats via a peripheral mechanism, and that endogenous serotonin has an inhibitory regulatory effect on gastric emptying in rats. Furthermore, the difference in rank order between the accelerating effect of gastric emptying and the 5-HT3 receptor antagonistic potencies in the cortex suggests that the 5-HT3-like receptor, modulating gastric emptying, is not identical to the classically defined 5-HT3 receptor.