5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (Isc) in guinea-pig isolated ileal mucosa over a wide concentration range (0.1 nM-0.1 mM). The concentration-response relationship was biphasic, consisting of a high potency phase (0.1 nM–1 μM) and a low potency phase (3–10 μM). Stimulation of Isc observed at the high potency phase tended to be sustained while responses at the low potency phase (3–10 μM) contained two components, an initial “transient” response followed by a “maintained” response. Both the high potency phase (maximum stimulation ∼30 μA cm−2) and the low potency phase (maximum stimulation ∼80 μA cm −2) 5-HT response were antagonized by tetrodotoxin (TTX, 0.3 μM) and atropine (1 μM). However, another low potency (3 μM-0.1 mM, maximum stimulation ∼30 μA cm−2) component of the 5-HT response was revealed in the presence of TTX or atropine. In the presence of methysergide (1 μM), the concentration-response relationship of 5-HT was still biphasic and tropisetron (0.1 and 10 μM) antagonized both phases of the 5-HT response. In the presence of methysergide, the high potency phase 5-HT response was mimicked by 5-methoxytryptamine (5-MeOT) and the selective 5-HT4 agonist SC-53116 but not by BIMU 8. The potent 5-HT4 antagonist GR 113808 antagonized the response to 5-MeOT in a surmountable manner with an affinity estimate of 9.6 ± 0.3 (n = 4). The 5-MeOT stimulated increase in Isc was also antagonized in an unsurmountable manner by granisetron (1 μM). In the presence of methysergide, desensitization of 5-HT3 receptors with 2-methyl-5-hydroxytryptamine (10 μM) abolished both phases of the 5-HT response. Under the same condition, desensitization of 5-HT4 receptors with 5-MeOT (10 μM) abolished only the high potency 5-HT response and dextrally shifted the low potency 5-HT response. These data show that neuronal and non-neuronal 5-HT receptors are involved in the regulation of secretion in ileal mucosa. We propose the presence of a neuronal 5-HT4 receptor located upstream of the well characterized neuronal 5-HT3 receptors to be responsible for the high potency 5-HT response. A schematic model is proposed to explain our findings and the relationship between this 5-HT4 receptor and other 5-HT receptor subtypes regulating secretion that have been described in the literature.