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 short-circuit current and DMBS in vitro; these responses were abolished by suramin (300 μM), a P2Y-receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB; 100 μM), a store-operated channel 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 U-73122 (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 store-operated channel.