To test for the role of the P2Y2 receptor(P2Y2-R) in the regulation of nucleotide-promoted Ca2+ signaling in the lung, we generated P2Y2-R- deficient (P2Y2-R(-/-)) mice and measured intracellular Ca2+(i) responses (ΔCa2+(i)) to nucleotides in cultured lung fibroblasts and nasal and tracheal epithelial cells from wild type and P2Y2-R(-/-) mice. In the wild type fibroblasts, the rank order of potencies for nucleotide-induced ΔCa2+(i) was as follows: UTP ≥ ATP >> ADP > UDP. The responses induced by these agonists were completely absent in the P2Y2R(-/-) fibroblasts. Inositol phosphate responses paralleled those of ACa2+(i) in both groups. ATP and UTP also induced Ca2+(i) responses in wild type airway epithelial cells. In the P2Y2-R(-/-) airway epithelial cells, UTP was ineffective. A small fraction (25%) of the ATP response persisted. Adenosine and α,β- methylene ATP were ineffective, and ATP responses were not affected by adenosine deaminase or by removal of extracellular Ca2+, indicating that neither P1 nor P2X receptors mediated this residual ATP response. In contrast, 2-methylthio-ADP promoted a substantial Ca2+(i) response in P2Y2-R(-/-) cells, which was inhibited by the P2Y1 receptor antagonist adenosine 3'-5'-diphosphate. These studies demonstrate that P2Y2-R is the dominant purinoceptor in airway epithelial cells, which also express a P2Y1 receptor, and that the P2Y2-R is the sole purinergic receptor subtype mediating nucleotide-induced inositol lipid hydrolysis and Ca2+ mobilization in mouse lung fibroblasts.