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]. To investigate whether functional 5-HT4 receptors are present in the equine small intestine. In vitro organ bath set up, applying electrical field stimulation (EFS) in longitudinal and circular smooth muscle strips. 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. 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.