The intracellular signaling pathways and molecular mechanisms responsible for P2-purinoceptor-mediated chloride (Cl-) currents (ICl,ATP) were studied in mouse ventricular myocytes. In standard NaCl containing extracellular solutions, extracellular ATP (100 µM) activated two different currents, ICl,ATP with a linear I-V relationship in symmetrical Cl- solutions, and an inwardly rectifying cation conductance (cationic IATP). Cationic IATP was selectively inhibited by Gd3+ and Zn2+, or by replacement of extracellular NaCl by NMDG; ICl,ATP was Cl- selective, and inhibited by replacement of extracellular Cl- by Asp-; both currents were prevented by suramin or DIDS pretreatment. In GTPS-loaded cells, ICl,ATP was irreversibly activated by ATP, but cationic IATP was still regulated reversibly. GDPS failed to activate the ICl,ATP, even though pertussis toxin pretreatment did not modulate ICl,ATP. These results suggest that activation of ICl,ATP occurs via a G-protein coupled P2Y purinergic receptor. The ICl,ATP persistently activated by GTPS, was inhibited by glibenclamide but not by DIDS, thus exhibiting known pharmacological properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In ventricular cells of cftr-/- mice, extracellular ATP activated cationic IATP, but failed to activate any detectable ICl,ATP. These results provide compelling evidence that activation of CFTR Cl- channels in mouse heart are coupled to G-protein coupled P2Y purinergic receptors.