Neuropeptides and their corresponding G protein-coupled receptors are increasingly implicated in the autocrine/paracrine stimulation of growth of human cancers. Using K-Ras mutated human pancreatic ductal adenocarcinoma cell line PANC-1 as a model system, we demonstrate that neurotensin (NT) induced translocation of phosphorylated extracellular signal-regulated kinases (ERK-1 and ERK-2) to the nucleus, rapid dose-dependent activation of dual-specificity mitogen and ERK-1 and ERK-2 kinase-1/2 (MEK-1/2), and striking stimulation of c-Raf-1 but not pan-Ras. Furthermore, treatment of PANC-1 cells with protein kinase C (PKC) inhibitors, GF-1 and Ro 31-8220, completely abrogated NT-induced ERK-1 and ERK-2 activation, and significantly attenuated NT-induced c-Raf-1 stimulation. Interestingly, NT did not stimulate epidermal growth factor receptor transactivation, and epidermal growth factor receptor tyrosine kinase or Src inhibitors did not affect NT-induced ERK activation in PANC-1 cells. Our results indicate that NT potently stimulates c-Raf-1-MEK-ERK in PANC-1 cells through a PKC-dependent signaling pathway. Furthermore, we show that NT-induced DNA synthesis in PANC-1 cells is ERK-dependent. Finally, we demonstrate that NT stimulated clonal growth of PANC-1 cells in semisolid medium, which is abrogated by both GF-1 and the MEK-1/2 inhibitor, U0126. Collectively our results suggest that PKC-mediated stimulation of ERK-1 and ERK-2 play a pivotal role in NT-induced growth of PANC-1 cells harboring activating K-Ras mutation.