Among the most important signaling pathways operating in pancreatic cancer cells are those resulting from mutations in the Ras oncogene or from overexpression of ErbB-2 and associated Src-family kinases. In this study, we aimed to characterize CHK expression and function in pancreatic cancer. Our data demonstrated CHK expression in human pancreatic cancer tissues, and also showed that CHK associated with ErbB-2 via its SH2 domain in human PANC-1 pancreatic cancer cells. PANC-1 cells were found to express both Src kinase and Lyn kinase, although the expression of Lyn kinase was more abundant. Furthermore, CHK downregulated Lyn kinase activity and significantly inhibited the in vitro growth and invasion of PANC-1 cells upon EGF stimulation. These results indicate that CHK is a negative regulator of ErbB-2 and Lyn kinase signaling in pancreatic cancer cells.