REASONS FOR PERFORMING STUDY: Selective 5-HT4 receptor agonists such as prucalopride are used as human prokinetics, since activation of 5-HT4 receptors on intestinal cholinergic neurons facilitates acetylcholine release. 5-HT4 receptors, linked to adenylyl cyclase, act via generation of cAMP. None of the 4 in vitro studies on 5-HT in horses provided evidence for neuronal 5-HT4 receptors, but none used the protocol as described in human studies [1-4]. OBJECTIVES: To investigate whether functional 5-HT4 receptors are present in the equine small intestine. STUDY DESIGN AND METHODS: In vitro organ bath set up, applying electrical field stimulation (EFS) in longitudinal and circular smooth muscle strips. RESULTS: Results were similar in both muscle layers. In the presence of 0.3 mmol/l NG-Nitro-L-arginine methyl ester and 0.3 µmol/l apamine, excluding effects of the inhibitory transmitters NO and ATP, EFS induced voltage-dependent on-contractions; these were neurogenic as they were abolished by 3 µmol/l tetrodotoxin. At a voltage inducing 50% of the maximal amplitude, the submaximal EFS-induced contractions were cholinergic as atropine (1 µmol/l) abolished them. Prucalopride (0.3 µmol/l) did not increase the amplitude of these submaximal EFS-induced contractions. Even in the presence of the nonselective phosphodiesterase inhibitor IBMX, previously shown to enhance the effect of neuronal 5-HT4 receptors by inhibiting breakdown of their 2nd messenger cAMP [5], prucalopride (3 µmol/l) had no influence. Also 5-HT (10 µmol/l), a full agonist at 5-HT4 receptors, tested in the presence of methysergide and granisetron to exclude interaction with other 5-HT receptor subtypes, did not enhance EFS-induced submaximal contractions. CONCLUSIONS: There is no evidence for presence of 5-HT4 receptors on the cholinergic neurons of the equine small intestine. These results question the application of 5-HT4 prokinetic drugs in horses. Ethical animal research: Research ethics committee oversight not currently required by this conference: the study was performed on material collected at an abattoir. Sources of funding: None. Competing interests: None declared.