The transcription factor nuclear factor (NF)-κB is activated by oxidative stress or cytokines and is critical to the activation of inflammatory genes. Here, we report that hydrogen peroxide or 3-morpholinosydnonimine, which simultaneously releases nitric oxide and superoxide, synergize with the cytokine tumor necrosis factor (TNF)-α to activate NF-κB in rat lung epithelial cells, suggesting that signaling pathways elicited by reactive oxygen species (ROS)/reactive nitrogen species (RNS) are different from TNF-induced signaling. These findings were substantiated by observations that levels of IκB-α did not change after exposure to ROS/RNS, whereas a rapid depletion of IκB-α was observed in cells exposed to TNF. In addition, the proteosome inhibitor MG132 did not affect activation of NF-κB by ROS/RNS, whereas it abolished the TNF response. Transfection of a dominant negative Ras construct prevented the activation of NF-κB by ROS/RNS, demonstrating the requirement for Ras in the activation of NF-κB by oxidants. In contrast, TNF activated NF-κB in a Ras-independent fashion. Evaluation of members of the mitogen-activated protein kinase (MAPK) family as downstream effectors of Ras revealed the requirement of MAPK/ extracellular-regulated kinase (ERK) kinase kinase (MEKK)1 and c-Jun N-terminal kinases in the induction of NF-κB by both oxidants and TNF, whereas the MEK-ERK pathway negatively regulates NF-κB. Our findings demonstrate that cytokines and oxidants cooperate in the activation of transcription factors through distinct pathways, and suggest that anti-inflammatory and antioxidant therapies may be required in concert to prevent the activation of NF-κB-regulated genes important in the development of inflammatory diseases.