5-HT(4) receptors mediate circular muscle relaxation in both human and canine large intestine, but this phenomenon alone can not explain the improvement in colonic motility induced by selective 5-HT(4) receptor agonists in vivo. We set out to characterize 5-HT(4) receptor-mediated effects in longitudinal muscle strips of canine and human large intestine. Electrical field stimulation (EFS) was applied providing submaximal isotonic contractions. L-NOARG (0.1 mM) was continuously present in the organ bath to preclude nitric oxide-induced relaxation to EFS. The selective 5-HT(4) receptor agonist prucalopride (0.3 microM) enhanced EFS-evoked contractions, that were antagonized in both preparations by the selective 5-HT(4) receptor antagonist GR 113808 (0.1 microM). The prucalopride-induced increase was present in canine ascending and descending colon, but absent in rectum. Regional differences in response to prucalopride were not observed in human ascending and sigmoid colon and rectum. Incubation with atropine (1 microM) or tetrodotoxin (0.3 microM) inhibited EFS-induced contractions, which were then unaffected by prucalopride (0.3 microM) in both tissues. In the presence of methysergide (3 microM; both tissues) and granisetron (0.3 microM; only human tissues), 5-HT (0.3 microM) enhanced EFS-induced contractions, an effect that was antagonized by GR 113808 (0.1 microM). In the presence of atropine or tetrodotoxin, EFS-induced contractions were inhibited, leaving 5-HT (0.3 microM) ineffective in both preparations. This study demonstrates for the first time that in human and canine large intestine, 5-HT(4) receptors are located on cholinergic neurones, presumably mediating facilitating release of acetylcholine, resulting in enhanced longitudinal muscle contractility. This study and previous circular muscle strip studies suggest that 5-HT(4) receptor agonism facilitates colonic propulsion via a coordinated combination of inhibition of circumferential resistance and enhancement of longitudinal muscle contractility.