Signal transduction pathways through protein kinase C (PKC) may play a significant role in DNA synthesis and proliferation of human pancreatic cancers. Treatment of human pancreatic ductal adenocarcinoma cell line PANC-1 with biologically active phorbol-12,13-dibutyrate led to striking activation of protein kinase D (PKD), a member of a novel family of serine/threonine kinases distinct from PKC isoforms. Using PANC-1 as a model system, we demonstrate that neurotensin (NT) induced a rapid and striking activation of PKD as determined by in vitro kinase assay and by in vivo phosphorylation of serines 744, 748, and 916. PKD activation induced by NT was abrogated by treatment of PANC-1 cells with PKC inhibitors GF-1 and Ro 31-8220. NT induced a rapid and transient translocation of PKD from the cytosol to the plasma membrane. Inhibiting PKC activity blocked the reverse translocation of PKD from the plasma membrane to the cytosol. Finally, we show that NT-induced DNA synthesis in PANC-1 cells is PKC-dependent. Collectively our results demonstrate, for the first time, the existence of a functional PKC/PKD signaling pathway in human ductal pancreatic carcinoma cells and suggest that PKCs mediate the mitogenic signaling process initiated by NT.