This study examines the mechanism of P2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (ISC). Both ATP-induced arachidonic acid (AA) synthesis and ISC in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclooxygenase inhibitor indomethacin decreased ISC and cAMP production in ATP-treated cells with an IC50 of ~0.3 µM. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and ISC evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and ISC triggered by ATP and AA but did not affect PKA activation and ISC detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca2+i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced ISC was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P2Y1 and P2Y2 than for other P2Y receptor subtypes. The rank order of potency (2MeSATP>ATP>ADP>>UTP) indicates that P2Y1 rather than P2Y2 receptors contribute to PKA and ISC activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.