1. Madin-Darby canine kidney (MDCK) cells are a widely used model system for the study of epithelial cells. We have utilized a clonal variant, MDCK-D1, to examine signalling by P2-purinoceptors. 2. Several lines of evidence that lead us to conclude that MDCK-D1 cells co-express P2a- and P2y-purinoceptors and that both subtypes are linked to the release of arachidonic acid and metabolites (AA) include: (a) relative potency of nucleotide analogues in promoting AA release; (b) blockade by the antagonist suramin of response to the P2Y-selective agonist, 2-methylthio ATP (2-MT-ATP), but not to the P2a-selective agonist, UTP; and (c) additivity of response to 2-MT-ATP and UTP. AA release is a consequence of activation of phospholipase A2 (PLA2), most likely the 85 kDa cytosolic PLA2. 3. Treatment of MDCK-D1 cells with ATP, but not UTP, increases inositol 1,4,5-trisphosphate formation while both UTP and ATP increase phosphatidylcholine hydrolysis, ATP, UTP, and 2-MT-ATP can also stimulate phospholipase D activity. 4. Purine nucleotides increase cellular cAMP levels in MDCK-D1 cells in a manner that depends, at least in part, on activation of cyclooxygenase, since cAMP generation stimulated by ATP or UTP is inhibited by treatment of cells with indomethacin. Because cyclooxygenase-derived PGE2 can bind to prostaglandin receptors and stimulate synthesis of cAMP, nucleotides may raise cAMP in an autocrine or paracrine fashion. 5. Taken together, these results indicate that MDCK-D1 cells co-express P2a and P2y-purinoceptors and that these receptors utilize several mechanisms to regulate cell function, including activation of multiple phospholipases and autocrine/paracrine action of products.