To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 μM) to block Na+channels, exposure of the apical membrane to ATP significantly increased the short-circuit current ( Isc) from 18.2 ± 5.9 to 52.8 ± 12.7 μA ( n = 18). The response to ATP is mediated by basolateral-to-apical Cl−transport because it is inhibited by 1) the Cl−channel blockers 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 μM) in the apical chamber, 2) the K+channel blocker Ba2+(5 mM), or 3) the Na+-K+-2Cl−cotransport inhibitor bumetanide (200 μM) in the basolateral chamber. Other nucleotides stimulated an increase in Iscwith a rank order potency of UTP = ATP = adenosine 5′-O-(3)-thiotriphosphate, consistent with P2upurinergic receptors. ADP, AMP, 2-methylthioadenosine 5′-triphosphate, and adenosine had no effect. A cDNA encoding a rat P2ureceptor (rP2uR) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl−secretion through activation of P2ureceptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.