Background & Aims: PGE2 plays a crucial role in the regulation of duodenal bicarbonate (HCO3-) secretion. We investigated the signal pathways involved in PGE2-mediated duodenal HCO3-secretion. Methods: Murine duodenal mucosal HCO3- secretion was examined in vitro in Ussing chambers in the presence of a variety of signal transduction modulators. Phosphatidylinositol-3 kinase (PI3K) activity was measured by immunoprecipitation of PI3K and ELISA. Results: PGE2 markedly stimulated duodenal HCO3-secretion. The cAMP-dependent signaling pathway inhibitors MDL-12330A and KT5720 reduced PGE2-stimulated HCO3-secretion by 23% and 20%, the Ca2+-influx inhibitor verapamil by 26%, the calmodulin antagonist W-13 by 24%, whereas the PI3K inhibitors wortmannin and LY294002 reduced PGE2-stimulated HCO3-secretion by 51% and 47%, respectively. Neither mitogen-activated protein kinase (MAPK) inhibitor PD98059 nor tyrosine kinase inhibitor genistein altered PGE2-stimulated HCO3- secretion. PGE2 application caused a rapid and concentration-dependent increase in duodenal mucosal PI3K activity. Conclusions: PGE2 stimulates duodenal mucosal HCO3- secretion via cAMP-, Ca2+-, and PI3K-dependent signaling pathways, and the PI3K pathway plays a major role in PGE2-mediated duodenal HCO3- secretion. This study has therefore revealed a novel signal transduction mechanism for the regulation of duodenal HCO3- secretion.