The composition of the fluid within the oviduct is largely determined by the secretory and absorptive activities of the oviduct epithelium. The present study explored the effects of basolateral nucleotide stimulation on ion transport in the bovine oviduct using the chamber short-circuit current technique. Basolateral application of ATP induced a rapid transient increase in ion secretion by oviduct epithelial monolayers in a concentration-de pendent manner. The ATP-induced short-circuit current (I-SC) response was preserved in the presence of amiloride, whereas it was reduced in the absence of extracellular chloride or in the presence of bumetanide. The channels underlying the chloride secretory response were identified as Ca2+-activated Cl- channels and CFTR. The ATP-induced Cl- secretory response was largely preserved in the absence of extracellular Ca2+ but was significantly reduced in the presence of BAPTA-AM (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid-acetomethoxy ester), thapsigargin, or 2-APB (2-aminoethoxydiphenylborate), demonstrating an important role for intracellular Ca2+ signaling in mediating these effects. A nucleotide potency profile of ATP = UTP (uridine triphosphate) > ADP, sensitivity to suramin, and cross-desensitization by basolateral UTP suggests that ATP exerted its effects on chloride secretion through the purinergic receptor P2Y, G protein-coupled 2, and the presence of the P2RY2 gene was confirmed by RT-PCR. These results provide strong evidence that purinergic signaling constitutes a key mechanism of regulating chloride secretion and thus fluid formation in the bovine oviduct.