Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, was originally purified from the rat stomach. Although ghrelin has been recognized as an important regulator of energy metabolism, the regulation of the ghrelin secretion is largely unknown. Here, we examined the direct effects of insulin, leptin, and glucagon on the release of ghrelin from the isolated rat stomach. The isolated pancreas-spleen-duodenum deprived preparation of rat stomach was used. After a baseline control infusion into the left gastric artery, insulin, leptin, or glucagon were infused for 15 min at concentrations of 0.1, 1, and 10 nM. The levels of immunoreactive ghrelin in the venous effluents were measured with a radioimmunoassay. Insulin and leptin inhibited ghrelin secretion dose-dependently (total amount of ghrelin release: insulin at 1 nM, 73.5+/-7.3% of the control infusion; leptin at 1 nM, 81.8+/-2.5% of the control infusion; n=5, P<0.05), while glucagon increased it dose-dependently (total amount of ghrelin released at 10 nM was 143.9+/-19.3% of the control infusion; n=5, P<0.01). These results indicate that the ghrelin responses observed in vivo could be due to direct effects of multiple hormonal signals on the stomach.