Increased arterial pressure, angiotensin II, and cytokines each result in feedback inhibition of renin gene expression. Because angiotensin II and cytokines can stimulate reactive oxygen species production, we tested the hypothesis that oxidative stress may be a mediator of this inhibition. Treatment of renin-expressing As4.1 cells with the potent cytokine tumor necrosis factor-α caused an increase in the steady-state levels of cellular reactive oxygen species, which was reversed by the antioxidant N-acetylcysteine. Exogenous H2O2 caused a dose- and time-dependent decrease in the level of endogenous renin mRNA and decreased the transcriptional activity of a 4.1-kb renin promoter fused to luciferase, which was maximal when the renin enhancer was present. The effect of H2O2 appeared to be specific to renin, because there was no change in the expression of β-actin or cyclophilin mRNA or transcriptional activity of the SV40 promoter. The tumor necrosis factor-α–induced decrease in renin mRNA was partially reversed by either N-acetylcysteine or panepoxydone, a nuclear factor κB (NFκB) inhibitor. Interestingly, H2O2 did not induce NFκB in As4.1 cells, and panepoxydone had no effect on the downregulation of renin mRNA by H2O2. The transcriptional activity of a cAMP response element-luciferase construct was decreased by both tumor necrosis factor-α and H2O2. These data suggest that cellular reactive oxygen species can negatively regulate renin gene expression via an NFκB-independent mechanism involving the renin enhancer and inhibiting cAMP response element–mediated transcription. Our data further suggest that tumor necrosis factor-α decreases renin expression through both NFκB-dependent and NFκB-independent mechanisms, the latter involving the production of reactive oxygen species.