Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y2 receptors stimulate luminal Ca2+-activated Cl- channels and inhibit Na+ transport. Here we address the mechanism of ATP-mediated inhibition of Na+ transport. M-1 cells had a transepithelial voltage (V(te)) of -31.4 +/- 1.3 mV and a transepithelial resistance (R(te)) of 1151 +/- 28 Omegacm(2). The amiloride-sensitive short circuit current (I(sc)) was -28.0 +/- 1.1 microA/cm2. The ATP-mediated activation of Cl- channels was inhibited when cytosolic Ca2+ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca2+](i) increase was paralleled by a rapid and transient R(te) decrease (297 +/- 51 Omegacm(2)). In the presence of CPA, basolateral ATP led to an R(te) increase by 144 +/- 17 Omegacm(2) and decreased V(te) from -31 +/- 2.6 to -26.6 +/- 2.5 mV. Isc dropped from -28.6 +/- 2.4 to -21.6 +/- 1.9 microA/cm2. Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na+ absorption. Lowering [Ca2+](i) by removal of extracellular Ca2+ did not alter the ATP effect. PKC inhibition or activation were without effect. Na+ absorption was activated by pH(i) alkalinization and inhibited by pH(i) acidification. ATP slightly acidified M-1 cells by 0.05 +/- 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y2 receptors inhibits Na+ absorption. This effect is not mediated via [Ca2+](i), does not involve PKC and is to a small part mediated via intracellular acidification.