Abstract 5-Hydroxytryptamine 4 (5-HT4) receptor agonists promote colonic propulsion. The alteration of circular muscle (CM) motility underlying this involves inhibition of contractility via smooth muscle 5-HT4 receptors and proximal colonic motility stimulation, the mechanism of the latter not having been characterized. Our aim was to identify and characterize a 5-HT4 receptor-mediated stimulation of human colon CM contractile activity. 5-HT4 receptor ligands were tested on electrical field stimulation (EFS)-induced contractions of human colonic muscle strips cut in the circular direction (called ‘whole tissue’ strips). Additionally, after incubation of tissues with [3H]-choline these compounds were tested on EFS-induced release of tritium in whole tissue strips and in ‘isolated’ CM strips, obtained by superficial cutting in the CM layer. Tetrodotoxin and atropine blocked EFS-induced contractions of whole tissue CM strips. Prucalopride (0.3 μmol L−1) evoked a heterogenous response on EFS-induced contraction, ranging from inhibition (most frequently observed) to enhancement. In the release experiments, EFS-induced tritium efflux was blocked by tetrodotoxin. Prucalopride increased EFS-induced tritium and [3H]-acetylcholine efflux in whole tissue and in isolated CM strips. All effects of prucalopride were antagonized by the selective 5-HT4 receptor antagonist GR113808. The results obtained indicate the presence of excitatory 5-HT4 receptors on cholinergic nerves within the CM of human colon.