Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long-term cellular activities but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1-5 min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser744 and Ser748 revealed striking PKC-independent phosphorylation of Ser748 as well as Ser744 phosphorylation that remained predominantly but not completely PKC-dependent, at later times of bombesin or vasopressin stimulation (20-90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser744 and Ser748 is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser748 is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.