It has been reported that the cystic fibrosis transmembrane conductance regulator (CFTR) can be activated through cAMP- and protein kinase A-independent pathways involving GTP-binding proteins and an unknown kinase. In this study, we further examined how G protein-coupled pathways regulate CFTR. We demonstrate that stimulation of purinergic P2Y(2) receptors in CFTR-expressing oocytes and in airway epithelial cells activates CFTR Cl(-) currents. Activation of CFTR Cl(-) currents via P2Y(2) was inhibited by CFTR(inh)-172 and was independent of intracellular Ca(2+), protein kinase C, or calmodulin-dependent kinase (CAMK). However, activation of CFTR was suppressed by inhibition of phospholipase C and by the nonselective protein kinase inhibitor staurosporine. Activation of CFTR through P2Y(2) receptors was enhanced when G(i) proteins were inhibited by pertussis toxin. Inhibition of protein kinase A and of protein kinases downstream of P2Y(2) receptors such as mitogen-activated protein kinases, tyrosine kinase, or c-src kinase did not interfere with activation of CFTR. The present results demonstrate an antagonistic regulation of CFTR by P2Y(2) receptors: CFTR is inhibited by stimulation of G(i) proteins and is activated by stimulation of G(q/11)/PLC and an unknown downstream protein kinase.