Airway epithelial cells are simultaneously exposed to and produce cytokines and reactive oxygen species (ROS) in inflammatory settings. The signaling events and the physiological outcomes of exposure to these inflammatory mediators remain to be elucidated. Previously we demonstrated that in cultured mouse lung epithelial cells exposed to bolus administration of H₂O₂, TNF-induced nuclear factor kappa B (NF-B) activity was inhibited, whereas c-Jun-N-terminal kinase (JNK), activation was enhanced via a mechanism involving TNF-receptor-1 (TNF-RI). In this study we utilized the non-phagocytic NADPH oxidase (Nox1), to study the effects of endogenously produced ROS on a line of mouse alveolar type II epithelial cells. Nox1 expression and activation inhibited TNF-induced inhibitor of kappa B kinase (IKK), and NF-B while promoting JNK activation and cell death. Nox1-induced JNK activation and cell death were attenuated through expression of a dominant negative TNF-RI construct, implicating a role for TNF-RI in Nox1 signaling. Furthermore, Nox1 utilized the TNF-RI adaptor protein TNF receptor associated factor-2 (TRAF2), and the redox regulated JNK MAP3K, apoptosis signal kinase-1 (ASK1), to activate JNK. Additionally, ASK1 siRNA attenuated both Nox1-induced JNK activity and cell death. Collectively, these studies suggest a mechanism in which ROS produced in lung epithelial cells activate JNK and cause cell death utilizing TNF-RI and the TRAF2-ASK1 signaling axis.