Since little is known about the role of P2Y receptors (purinoceptors) in duodenal mucosal bicarbonate secretion (DMBS), we sought to investigate the expression and function of these receptors in duodenal epithelium. Expression of P2Y₂ receptors was detected by RT-PCR in mouse duodenal epithelium and SCBN cells, a duodenal epithelial cell line. UTP, a P2Y₂ receptor agonist, but not ADP (10 µM) significantly induced murine duodenal I_sc and DMBS in vitro; these responses were abolished by suramin (300 µM), a P2Y receptor antagonist, or 2-APB (100 µM), a store-operated channel (SOC) blocker. Mucosal or serosal addition of UTP induced a comparable DMBS in wild-type mice but markedly impaired response occurred in P2Y₂ knockout mice. Acid-stimulated DMBS in vivo was significantly inhibited by suramin (1 mM) or PPADS (30 µM). Both ATP and UTP, but not ADP (1 µM), raised cytoplasmic free Ca²⁺ concentrations ([Ca²⁺]cyt) with similar potencies in SCBN cells. ATP-induced [Ca²⁺]cyt was attenuated by U73122 (10 µM), La³⁺ (30 µM), or 2-APB (10 µM) but was not significantly affected by nifedipine (10 µM). UTP (1 µM) induced a [Ca²⁺]_cyt transient in Ca²⁺-free solutions and restoration of external Ca²⁺ (2 mM) raised [Ca²⁺]_cyt due to capacitative Ca²⁺ entry. La³⁺ (30 µM), SK&F96365 (30 µM) and 2-APB (10 µM) inhibited UTP-induced Ca²⁺ entry by 92%, 87% and 94%, respectively. Taken together, our results imply that activation of P2Y₂ receptors enhances DMBS via elevation of [Ca²⁺]_cyt that likely results from an initial increase in intracellular Ca²⁺ release followed by extracellular Ca²⁺ entry via SOC.