Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (InsP_x) accumulation and Ca²⁺ mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [³H]InsP_x accumulation and Ca²⁺ mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y₁ receptors, N⁶-cyclopentyladenosine and AMP, ,-methylene-ATP and ,-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [³H]InsP_x accumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [³H]InsP_x accumulation and Ca²⁺ mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [³H]InsP_x accumulation and Ca²⁺ mobilization in canine TECs. P2Y₂ receptors may be predominantly mediating [³H]InsP_x accumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca²⁺ mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.