The importance of reactive oxygen species (ROS) or changes in cellular redox state in signal transduction and gene regulation is becoming increasingly evident. In this study, we tested the hypothesis that ROS are directly involved in the induction of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) and mediate the induction of MnSOD by tumor necrosis factor-alpha (TNF-alpha). Pretreatment of human pulmonary adenocarcinoma cells H441 with the antioxidants N-acetyl-L-cysteine (NAC) and nordihydroguaiaretic acid (NDGA) blocked MnSOD induction by TNF-alpha, implicating ROS as a signaling agent in this pathway. Treatment of H441 cells with the exogenous oxidants hydrogen peroxide (H2O2) and diamide increased MnSOD mRNA, supporting the hypothesis that ROS directly affect expression of MnSOD. The temporal pattern of MnSOD induction differed for TNF-alpha and H2O2, suggesting distinct signaling pathways. DNA binding of two redox-sensitive transcription factors, NF-kappa B and activator protein (AP)-1, was evaluated. TNF-alpha increased nuclear factor (NF)-kappa B-DNA binding, an effect blocked by pretreatment with NAC. H2O2 did not alter NF-kappa B-DNA binding. There was no evidence of AP-1 binding in cells treated with either TNF-alpha or H2O2. We conclude that ROS directly alter MnSOD expression and are involved in the induction of MnSOD by TNF-alpha.