One reason why pancreatic cancer is so aggressive and unresponsive to treatments is its resistance to apoptosis. We report here that reactive oxygen species (ROS) are a prosurvival, antiapoptotic factor in pancreatic cancer cells. Human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cells generated ROS, which was stimulated by growth factors (serum, insulin-like growth factor I, or fibroblast growth factor-2). Growth factors also stimulated membrane NAD(P)H oxidase activity in these cells. Both intracellular ROS and NAD(P)H oxidase activity were inhibited by antioxidants tiron and N-acetylcysteine and the inhibitor of flavoprotein-dependent oxidases, diphenylene iodonium, but not by inhibitors of various other ROS-generating enzymes. Using Rho(0) cells deficient in mitochondrial DNA, we showed that a nonmitochondrial NAD(P)H oxidase is a major source of growth factor-induced ROS in pancreatic cancer cells. Among proteins that have been implicated in NAD(P)H oxidase activity, MIA PaCa-2 and PANC-1 cells do not express the phagocytic gp91(phox) subunit but express several nonphagocytic oxidase (NOX) isoforms. Transfection with Nox4 antisense oligonucleotide inhibited NAD(P)H oxidase activity and ROS production in MIA PaCa-2 and PANC-1 cells. Inhibiting ROS with the antioxidants, Nox4 antisense, or MnSOD overexpression all stimulated apoptosis in pancreatic cancer cells as measured by internucleosomal DNA fragmentation, phosphatidylserine externalization, cytochrome c release, and effector caspase activation. The results show that growth factor-induced ROS produced by NAD(P)H oxidase (probably Nox4) protect pancreatic cancer cells from apoptosis. This mechanism may play an important role in pancreatic cancer resistance to treatment and thus represent a novel therapeutic target.