1. Extracellular nucleotides modulate ionic transport mechanisms in various epithelia. In the present study, we investigated the effects of extracellular ATP on the intracellular free Ca+2 concentration ([Ca2+]i) and electrophysiological properties of Necturus maculosus proximal convoluted tubule (PCT). 2. ATP raised [Ca2+]i in microdissected fura-2-loaded PCTs (half-maximal effect, approximately mumol 1(-1) ATP). The initial ATP-induced changes in [Ca2+]i were not blunted by the removal of external Ca2+ nor by the presence of Ca2+ channel blockers, but were abolished by thapsigargin and suramin. The sequence for the potency of various agonists on [Ca2+]i was 2-methylthioATP (2MeSATP) = ADP = ATP >> UTP, 2',3',-O-(4-benzoilbenzoil) ATP (BzATP), alpha, beta-methylene ATP (AMPCPP), adenosine. 3. In vivo electrophysiological measurements showed that 100 mumol 1(-1) peritubular ATP added to a Ringer solution reduced the basolateral cell membrane potential (Vm) and increased the cell membrane input conductance. In a low Cl- solution, this ATP-induced depolarization was enhanced. These effects were inhibited by 1 mmol l-1 SITS, consistent with the activation of a basolateral Cl- conductance. 4. The ATP-induced change in Vm was reproduced by ADP but not by UTP or adenosine, and was prevented by suramin. 5. The ATP-induced membrane depolarization was not influenced by thapsigargin, BAPTA AM, or staurosporine and was not reproduced by manoeuvres increasing [Ca2+]i or intracellular cAMP content. 6. We conclude that, in Necturus PCT, a P2y receptor mobilizes Ca2+ mainly from intracellular pools and increases a basolateral Cl- conductance, GCl. The activation of GCl occurs by a mechanism which is not related either to an increase in [Ca2+]i or cAMP content, or to PKC activation.