To test for the role of the P2Y(2) receptor (P2Y(2)-R) in the regulation of nueleotide-promoted Ca2+ signaling in the lung, we generated P2Y(2)-R-deficient (P2Y(2)-R(-/-)) mice and measured intracellular Ca-i(2+) responses (Delta Ca-i(2+)) to nucleotides in cultured lung fibroblasts and nasal and tracheal epithelial cells from wild type and P2Y(2)-R(-/-) mice. In the wild type fibroblasts, the rank order of potencies for nucleotide-induced Delta Ca-i(2+) was as follows: UTP greater than or equal to ATP much greater than ADP > UDP. The responses induced by these agonists were completely absent in the P2Y(2)-R(-/-) fibroblasts. Inositol phosphate responses paralleled those of Delta Ca-i(2+) in both groups. ATP and UTP also induced Ca-i(2+) responses in wild type airway epithelial cells. In the P2Y(2)-R(-/-) airway epithelial cells, UTP was ineffective, A small fraction (25%) of the ATP response persisted. Adenosine(-) and alpha,beta-methylene ATP were ineffective, and ATP responses were not affected by adenosine deaminase or by removal of extracellular Ca-i(2+) indicating that neither P1 nor P2X receptors mediated this residual ATP response. In contrast, 2-methylthio-ADP promoted a substantial Ca-i(2+) response in P2Y(2)-R(-/-) cells, which was inhibited by the P2Y(1) receptor antagonist adenosine 3'-5'-diphosphate. These studies demonstrate that P2Y(2)-R is the dominant purinoceptor in airway epithelial cells, which also express a P2Y(1) receptor, and that the P2Y(2)-R is the sole purinergic receptor subtype mediating nucleotide-induced inositol lipid hydrolysis and Ca2+ mobilization in mouse lung fibroblasts.