Pancreas cancer is the fourth leading cause of cancer-related death in adults in the United States. New molecular targets for diagnosis and therapy of this disease are desperately needed. In this study, we report on the mitotic serine-threonine kinase polo-like kinase 1 (Plk1) in pancreatic cancer. Plk1 mRNA was found to be overexpressed in 9 of 10 tested pancreatic cancer cell lines and in 4 of 4 tested human tumors. Immunohistochemical staining of a pancreatic tissue microarray showed that 26 of the 35 tumors taken directly from patients overexpressed Plk1. We also examined the effects of depleting Plk1 in pancreatic cancer cells by the use of antisense oligonucleotides. Antisense-treated pancreatic cancer cells showed cell cycle arrest in G(2)-M as well as a drastic reduction in proliferation rates. These data suggest that Plk1 is a potential therapeutic target in devising a treatment for patients with pancreatic cancer.