Extracellular nucleotides regulate renal transport. A luminal P2Y2 receptor in mouse cortical collecting duct (CCD) principal cells has been demonstrated elsewhere. Herein the effects of adenosine triphosphate (ATP) and uridine triphosphate (UTP) on electrogenic Na+ absorption in perfused CCD of mice kept on a low-NaCl diet were investigated. Simultaneously, transepithelial voltage (V(te)), transepithelial resistance (R(te)), and fura-2 [Ca2+]i fluorescence were measured. Baseline parameters were V(te), -16.5 +/- 1.2 mV; R(te), 80.8 +/- 7.1 Omega cm2; and equivalent short-circuit current (I(sc)), -261.0 +/- 25.1 microA/cm2 (n = 45). Amiloride (10 microM) almost completely inhibited I(sc) to -3.9 +/- 3.8 microA/cm2 (n = 10). Luminal ATP (100 microM) reduced V(te) from -16.5 +/- 2.1 to -12.5 +/- 1.93 and increased R(te) from 113.1 +/- 16.2 to 123.8 +/- 16.7 Omega cm2, which resulted in a 31.7% inhibition of amiloride-sensitive I(sc) (n = 12). Similarly, luminal UTP reversibly reduced V(te) from -22.0 +/- 2.1 to -13.6 +/- 2.1 mV and increased R(te) from 48.4 +/- 5.3 to 59.2 +/- 7.1 Omega cm2, which resulted in 49.1% inhibition of Na+ absorption (n = 6). In parallel, luminal ATP and UTP elevated [Ca2+]i in CCD, increasing the fura-2 ratio by 2.7 +/- 0.7 and 4.0 +/- 1.2, respectively. Basolateral ATP and UTP (100 microM) also inhibited amiloride-sensitive I(sc) by 21.8 (n = 14) and 20.1% (n = 8), respectively. Inhibition of luminal nucleotide-induced [Ca2+]i increase by Ca2+ store depletion with cyclopiazonic acid (3 microM) did not affect nucleotide-mediated inhibition of Na+ transport (n = 7). No evidence indicated the activation of a luminal Ca2+-activated Cl- conductance, a phenomenon previously shown in M-1 CCD cells (J Physiol 524: 77-99, 2000). In essence, these data indicate that luminal ATP and UTP, most likely via P2Y2 receptors, mediate inhibition of amiloride-sensitive I(sc) in perfused mouse CCD. This inhibition appears to occurs independently of an increase of cytosolic Ca2+.