During pancreatic tumorigenesis, the equilibrium between cell survival and cell death is altered, allowing aggressive neoplasia and resistance to radiation and chemotherapy. Local oxidative stress is one mechanism regulating programmed cell death and growth and may contribute to both tumor progression and suppression. Our recent in situ immunohistochemical studies demonstrated that levels of total nitrotyrosine, a footprint of the reactive nitrogen species peroxynitrite, are elevated in human pancreatic ductal adenocarcinomas. In this study, quantitative HPLC-EC techniques demonstrated a 21- to 97-fold increase in the overall levels of nitrotyrosine of human pancreatic tumor extracts compared to normal pancreatic extracts. Western blot analysis of human pancreatic tumor extracts showed that tyrosine nitration was restricted to a few specific proteins. Immunoprecipitation coupled with Western analysis identified c-Src tyrosine kinase as a target of both tyrosine nitration and tyrosine phosphorylation. Peroxynitrite treatment of human pancreatic carcinoma cells in vitro resulted in increased tyrosine nitration and tyrosine phosphorylation of c-Src kinase, increased (>2-fold) c-Src kinase activity, and increased association between c-Src kinase and its downstream substrate cortactin. Collectively, these observations suggest that peroxynitrite-mediated tyrosine nitration and tyrosine phosphorylation of c-Src kinase may lead to enhanced tyrosine kinase signaling observed during pancreatic ductal adenocarcinoma growth and metastasis.