We aimed to study 5‐HT4 receptors in canine stomach contractility both in vivo and in vitro. In anaesthetized Beagle dogs, the selective 5‐HT4 receptor agonist prucalopride (i.v.) induced dose‐dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the selective 5‐HT4 receptor antagonist GR 125487 (10 μg kg−1, i.v.). Electrical field stimulation (EFS) of corpus longitudinal muscle strips resulted in atropine‐ and tetrodotoxin‐sensitive contractions (L‐NOARG (0.1 mM) present in all organ bath solutions). Prucalopride increased these contractions (maximal response after single‐dose addition (0.3 μM): 165% of initial value, or after cumulative addition: 188%). In the presence of methysergide (3 μM), 5‐HT also increased EFS‐contractions (after single‐dose addition (0.3 μM): increase to 192%, after cumulative addition: 148%). The selective 5‐HT4 receptor antagonists GR 113808 (0.1 μM) or GR 125487 (10 nM) antagonized the prucalopride (0.3 μM)‐induced contraction increments. When EFS‐induced contractions were blocked by atropine or tetrodotoxin, prucalopride was ineffective. In the presence of methysergide (3 μM), the contraction increases to 5‐HT (0.3 μM) were prevented by GR 113808 (0.1 μM). The prucalopride curve (pEC50 7.9) was shifted in parallel to the right by GR 113808 3 nM (pA2 9.4). In the presence of methysergide (3 μM), the curve to 5‐HT (pEC50 8.1) was competitively antagonized by GR 113808, yielding a Schild slope of 0.8±0.2 (pKB of 9.1 with unit Schild slope). In corpus circular muscle strips, the prucalopride (0.3 μM)‐induced augmentation of EFS‐contractions (258%) was also prevented by GR 113808 (0.1 μM) (124%). In conclusion, the effects of 5‐HT4 receptor agonists on proximal stomach motor activity in vivo can be explained by an effect on 5‐HT4 receptors on cholinergic nerves within the gastric muscle wall. British Journal of Pharmacology (2001) 132, 1941–1947; doi:10.1038/sj.bjp.0703985