The intracellular signaling pathways responsible for extracellualr uridine-5'-triphosphate (UTP_o)-induced chloride (Cl⁻) currents (I_Cl.UTP) were studied in mouse ventricular myocytes with the whole-cell clamp technique. UTP_o (0.1 to 100 μM) activated a whole-cell current that showed a time-independent activation, a linear current-voltage relationship in symmetrical Cl⁻ solutions, an anion selectivity of Cl⁻ > iodide > aspartate, and an inhibition by a thiazolidinone-derived specific inhibitor (CFTR_inh-172, 10 μM) of cystic fibrosis transmembrane conductance regulator (CFTR), but not by a disulfonic stilbene derivative (DIDS, 100 μM), these properties matching those of CFTR Cl⁻ channels. The potency order of nucleotides for an activation of the Cl⁻ current was UTP = ATP > uridine-5'-diphosphate (UDP) = ADP. Suramin (100 μM), a P2Y receptor antagonist, strongly inhibited the UTP_o-activation of the Cl⁻ current, whereas pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 μM), another P2Y receptor antagonist, induced little inhibition of I_Cl.UTP. The activation of I_Cl.UTP was sensitive to protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, intracellular GDPβS (nonhydrolyzable GDP analogue) or anti-Gq/11 antibody. UTP_o failed to activate the Cl⁻ current when the cells were dialyzed with nonhydrolyzable ATP analogues (ATPS or AMP-PNP) without ATP, suggesting that ATP hydrolysis is a prerequisite for the current activation. I_Cl.UTP was persistently activated with a mixture of ATPγS + ATP in the pipette, suggesting the involvement of phosphorylation reaction in the current activation process. Our results strongly suggest that I_Cl.UTP is due to the activation of CFTR Cl⁻ channels through Gq/11-coupled P2Y₂ receptor-PLC-PKC signaling and ATP hydrolysis in mouse heart.