Protein kinase D (PKD), also called protein kinase Cμ (PKCμ), is a serine/threonine kinase that has unique enzymic and structural properties distinct from members of the PKC family of proteins. In freshly isolated rat parotid acinar salivary cells, extracellular ATP rapidly increased the activity and phosphorylation of PKD. The stimulation by ATP required high concentrations, was mimicked by the P2X₇ receptor ligand BzATP [2′- and 3′-O-(4-benzoylbenzoyl)ATP], and was blocked by Mg²⁺ and 4,4′-di-isothiocyano-2,2′-stilbene disulphonate (DIDS), suggesting that activation of PKD was mediated by P2X₇ receptors, which are ligand-gated non-selective cation channels. Phorbol ester (PMA) and the activation of muscarinic and substance P receptors also increased PKD activity. PKC inhibitors blocked ligand-dependent PKD activation and phosphorylation, determined by in vitro phosphorylation studies and by phospho-specific antibodies to two activation loop sites (Ser⁷⁴⁴ and Ser⁷⁴⁸) and an autophosphorylation site (Ser⁹¹⁶). ATP and BzATP also increased the tyrosine phosphorylation and activity of PKCΔ, and these stimuli also increased extracellular signal-regulated protein kinase (ERK) 1/2 activity in a PKC-dependent manner. PKD activation was not promoted by pervanadate (an inhibitor of tyrosine phosphatases) and was not blocked by PP1 (an inhibitor of Src family kinases) or genistein (a tyrosine kinase inhibitor), suggesting that tyrosine kinases and phosphatases did not play a major role in PKD activation. P2X₇ receptor-mediated signalling events were not dependent on Ca²⁺ entry. These studies indicate that PKC is involved in cellular signalling initiated by P2X₇ receptors as well as by G-protein-coupled receptors, and demonstrate that PKD and ERK1/2 are activated in similar PKC-dependent signalling pathways initiated by these diverse receptor types.