Endogenous ghrelin causes interdigestive contractions of the stomach in rats. In contrast, previous studies showed that 5-HT₃ and 5-HT₄ receptors were involved in regulating intestinal interdigestive contractions. We studied the possible role of endogenous ghrelin and 5-HT regulating interdigestive gastrointestinal (GI) contractions in rats. Four strain gauge transducers were implanted on the antrum, duodenum, and proximal and distal jejunum. After an overnight fast, GI contractions were recorded in freely moving conscious rats and ghrelin receptor antagonists [(D-lys3)GHRP6; 1 µmol/kg], 5-HT₃ antagonists (Ondansetron; 0.5 mg/kg) and 5-HT₄ antagonists (GR 125,487; 1 mg/kg) were administered (bolus, iv). To evaluate the relationship between the luminal concentrations of 5-HT and phase III-like contractions of the duodenum, duodenal juice was collected via the intraduodenal catheter. 5-HT content of the duodenal juice was measured by HPLC. (D-lys3)GHRP6 significantly attenuated the occurrence and amplitude of phase III-like contractions of the antrum, but not the duodenum and jejunum. 5-HT₄ antagonists significantly reduced spontaneous phase III-like contractions of the jejunum, without affecting those of the antrum and duodenum. In contrast, 5-HT₃ antagonists did not affect phase III-like contractions in GI tract. Luminal concentration of 5-HT at the phase III-like contraction (36.0 ±13.3 ng/ml, n=9) was significantly higher than that at the phase I-like contractions of the duodenum (4.9 ±1.6 ng/ml, n=9, P<0.05). It is suggested that released ghrelin from the gastric mucosa mediates gastric phase III-like contractions, while 5-HT released from enterochromaffin cells of the duodenal mucosa mediates intestinal phase III-like contractions via 5-HT₄receptors.