Previously, we presented in vivo evidence for a physiological significance of cAMP-regulated CFTR Cl(-) channels in Ca(2+)-activated Cl(-) reabsorption in the ductal system of the rat submandibular gland. Here, we address the mechanism by which basal CFTR activation contributes to the transepithelial Cl(-) movement evoked by muscarinic stimulation. The Cl(-) concentration ([Cl(-)]) was increased in the final saliva from rat submandibular gland during pilocarpine stimulation when a small interfering RNA for CFTR or a specific CFTR inhibitor, CFTR(inh)-172, was injected retrogradely into the gland's own duct, indicating that basal CFTR activation is involved in Cl(-) reabsorption. Systemically administered propranolol failed to alter the [Cl(-)], suggesting little involvement of a beta-adrenergic pathway in the Cl(-) movement that occurs through basal CFTR activation. Intraductal injection of suramin (a nonspecific P2-receptor antagonist) increased the salivary [Cl(-)], indicating the existence of endogenous purinergic activation. Upon separate intraductal injection, ATP and a P2Y(2)-receptor agonist, UTP, decreased the salivary [Cl(-)] almost equipotently. CFTR(inh)-172 and suramin each prevented these effects, whereas 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), a P2X(7) agonist, had no specific effect. Pilocarpine stimulation evoked ATP secretion into the salivary fluid. Immunohistochemistry revealed the partial coexistence of CFTR and P2Y(2) receptors on the luminal surface of epithelial cells in the striated ducts of this gland. These results raise the possibility that muscarinic stimulation-induced Cl(-) reabsorption occurs through basal CFTR activity and that this is regulated by P2Y(2) receptors in the ductal epithelium via stimulation by ATP secreted into the salivary fluid.