Extracellular nucleotides regulate renal ion transport. With the use of in vitro perfusion and [Ca(2+)](i) imaging, this study investigated whether mouse and rabbit cortical collecting ducts (CCD) respond to luminal nucleotides. In mouse CCD, luminal ATP (EC(50): 10 microM) and UTP (EC(50): 9.7 microM) increased [Ca(2+)](i) with an initial peak and a plateau. To make certain that basolateral P2 receptors were not activated by luminal nucleotides via leak diffusion, luminal trypsin (1 microM), a known agonist for basolateral proteinase-activated receptors, was perfused. Mouse CCD that were responsive to luminal ATP were nonresponsive to luminal trypsin but always showed [Ca(2+)](i) elevations by basolateral trypsin (10 or 100 nM). Luminal alpha,beta- and beta,gamma-methylene ATP, 2-methyl-S-ATP, ADP, UDP, and 2',3'-O-4-benzoylbenzoyl ATP had no effect (100 microM, n = 9). Without external Ca(2+), luminal ATP still stimulated a [Ca(2+)](i) increase. Mouse CCD also responded to basolateral ATP (EC(50): 23 microM) and UTP (EC(50): 23 microM) with smaller [Ca(2+)](i) elevations. Confocal microscopy of perfused CCD showed that luminal ATP (100 microM) rapidly increased [Ca(2+)](i) in nearly all cells (n = 6) and the same cells that responded to luminal ATP responded to basolateral ATP (100 microM). In contrast, rabbit CCD did not respond to luminal ATP/UTP (n = 8) despite ATP's known effect from the basolateral side (EC(50): 34 microM). These data indicate the expression of luminal P2Y receptors (probably P2Y(2)) in principal cells of mouse CCD but not in rabbit CCD.