Previously, we presented in vivo evidence for a physiological significance of cAMP-regulated CFTR Cl^- channels in Ca²⁺-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 (siRNA) 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 -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 P2Y₂-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 P2X₇ agonist, had no specific effect. Pilocarpine stimulation evoked ATP secretion into the salivary fluid. Immunohistochemistry revealed the partial coexistence of CFTR and P2Y₂ 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 P2Y₂ receptors in the ductal epithelium via stimulation by ATP secreted into the salivary fluid.