Protein kinase D (PKD), a novel serine-threonine kinase which acts downstream of PKC isoforms, has been implicated in important intracellular functions, such as cell survival and secretion. Recently, we have demonstrated a critical role for PKD in stimulated peptide secretion from the BON endocrine cell line; the role of PKD in pancreatic acinar cell secretion is not known. The purpose of our study was to: 1) delineate the expression and activation of PKD1 with secretagogue (i.e., CCK)-induced acinar cell secretion, and 2) define the upstream regulation of PKD1 activity by various PKC isoforms. Methods: Pancreatic acinar cells were isolated from female Swiss-Webster mice, and expression and localization of PKD1 was assessed by Western blot and immunofluorescent staining, respectively. PKD1 activation was determined by Ser744/748 phosphorylation and by translocation assays. Pancreatic acinar cells were treated with either CCK or caerulein (100 pM to 10 nM), with or without the PKC inhibitors GF-109293X (GFX; a general PKC inhibitor) or Gö-6976 (which inhibits PKC-α and PKC-β1) to determine the role of PKC isoforms in PKD1 activation and associated amylase secretion. Results: PKD1 is expressed throughout the acinar cell cytoplasm and plasma membrane. PKD1 phosphorylation was detected at 5 min after treatment with 10 nM CCK, peaked at 30 min, and diminished to basal levels after 3 h. CCK-induced PKD1 activation and amylase release were decreased by GFX but not Gö-6976, suggesting the involvement of PKC isoforms other than PKC-α and PKC-β1. Conclusions: Importantly, our data are the first to demonstrate PKD1 activation in pancreatic acinar cells following treatment with CCK. This activation is mediated, directly or indirectly, through upstream PKC isoforms. Moreover, our results suggest a role for PKD1 activation in pancreatic acinar cell enzyme secretion.