The transcription factor NF-B plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKC and are key regulators of NF-B activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-, and ethanol. However, the downstream participants in regulating NF-B activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKC and PKC in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh); and that PKD1 is necessary for NF-B activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose-dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-B activity stimulated by CCK-8 or CCh, as measured by NF-B DNA binding. Either inhibition of PKC or by isoform-specific inhibitory peptides; genetic deletion of PKC and in pancreatic acinar cells; or knockdown of PKD1 by using siRNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-B activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-B activation. Finally, the kinetics of PKD1 and NF-B activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-B activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKC and in the signaling pathways mediating NF-B activation in pancreatitis.