Ghrelin, des-acyl ghrelin, and obestatin are derived from a common prohormone, preproghrelin by posttranslational processing, originating from endocrine cells in the stomach. To examine the regulatory roles of these peptides, we applied the manometric measurement of gastrointestinal motility in freely moving conscious rat or mouse model. Ghrelin exerts stimulatory effects on the motility of antrum and duodenum in both fed and fasted state of animals. Des-acyl ghrelin exerts inhibitory effects on the motility of antrum but not on the motility of duodenum in the fasted state of animals. Obestatin exerts inhibitory effects on the motility of antrum and duodenum in the fed state but not in the fasted state of animals. NPY Y2 and Y4 receptors in the brain may mediate the action of ghrelin, CRF type 2 receptor in the brain may mediate the action of des-acyl ghrelin, whereas CRF type 1 and type 2 receptors in the brain may mediate the action of obestatin. Vagal afferent pathways might be involved in the action of ghrelin, but not involved in the action of des-acyl ghrelin, whereas vagal afferent pathways might be partially involved in the action of obestatin.