Apical and/or basolateral membranes of polarized epithelia express P2Y receptors which regulate the transport of fluids and electrolytes (Bucheimer and Linden, 2004). In the airway, P2Y receptors modulate Cl- secretion and Na+ reabsorption through the phospholipase C (PLC) and calcium signaling pathways. In airway epithelium, P2Y2 is the predominant receptor which classically couples to PLC and Ca2+ signaling cascade which enables ATP and UTP to increase intracellular calcium concentrations ([Ca2+]i) (Nicholas et al., 1996). The functional expression of other P2Y receptor subtypes, in particular the presence and function of apical versus basolateral P2Y6 receptors in airway epithelium remains controversial. In addition, other Ca2+-independent pathway(s) for P2Y6 receptor-mediated Cl- secretion have been proposed in human airway epithelia including the involvement of the cAMP-dependent cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel (Dulong et al., 2007; Wong et al., 2009). In this study, we examined P2Y receptor subtype expression, including P2Y6, and the effect of extracellular nucleotides on basal short-circuit current (ISC), intracellular calcium concentration ([Ca2+]i) and real-time changes in [cAMP] in a human bronchial epithelial cell line (16HBE14o-). Real-time PCR and Western blot analysis demonstrated P2Y1, P2Y2, P2Y4 and P2Y6 receptor expression. Using a simultaneous measurement technique of bioelectric and fluorescent signals, it was found that P2Y6 receptor agonist, UDP, stimulated a concomitant increase in ISC and [Ca2+]i in a concentration-dependent manner. The increase in ISC was inhibited by a specific CFTR inhibitor, CFTRinh-172. UDP stimulated an increase in PKA activity when measured by PepTag® non-radioactive cAMP-dependent protein kinase assay (Promega), suggesting that P2Y6 receptor might be coupled to CFTR-dependent Cl- secretion via cAMP/PKA pathways. To further confirm that UDP could increase cAMP level in 16HBE14o- cells, real-time cAMP changes in living cells were monitored by using CFP-Epac-YFP, an Epac-based polypeptide FRET reporter (van der Krogt et al., 2008). In 16HBE14o- cells expressing the CFP-Epac-YFP indicator, the cAMP elevating agent, forskolin, or UDP evoked an increase in CFP/FRET emission ratio, which indicated an increase in intracellular cAMP level. These data therefore suggest that activation of P2Y6 receptors by UDP stimulates real-time increase in [Ca2+]i and [cAMP], and that both Ca2+ and cAMP/PKA signaling pathways are important in regulating P2Y6 receptor-coupled ion transport in human bronchial epithelia.