Experiments were performed to characterize the P2 purinoceptor subtype responsible for cytoplasmic calcium mobilization in cells from the initial part of rabbit distal convoluted tubule (DCT). Free calcium concentration was measured in a DCT cell line (DC1) with the probe fura 2. Both ATP and UTP increased cytosolic Ca21 concentration ([Ca21]i; EC50 3 and 6 mM, respectively). The order of potency for nucleotide analogs was ATP 5 UTP . adenosine 59-O-[thiotriphosphate] .. ADP . UDP, which is consistent with the pharmacology of the P2Y2 receptor subtype. The increased [Ca21]i responses to ATP and UTP were strongly inhibited by suramin. Pretreatment of cells with pertussis toxin (PTX) attenuated the action of both nucleotides. Inhibition of phospholipase C with U-73122 totally blocked the [Ca21]i response to ATP. Thus ATP- and UTP-stimulated [Ca21]i mobilization in DC1 cells appears to be mediated via the activation of P2Y2 purinoceptors coupled to a G protein mechanism that is partially sensitive to PTX. Calcium flux measurements showed that lanthanum- and nifedipine-sensitive calcium channels are involved in the [Ca21]i responseto ATP.