GADD153 is a CCAAT/enhancer-binding-protein-related gene that may function to control cellular growth in response to stress signals. In this study, a variety of oxidant treatments were shown to stimulate endogenous GADD153 mRNA expression and to transcriptionally activate a GADD153 promoter–reporter gene construct in transfected HeLa cells. Both commonalities and distinctions in the induction of GADD153 by H₂O₂ and the thiol-reactive compound arsenite were demonstrated. GADD153 mRNA induction by both H₂O₂ and arsenite was potentiated by GSH depletion, and completely inhibited by N-acetyl-cysteine. o-Phenanthroline and mannitol blocked GADD153 induction by H₂O₂, indicating that iron-generated hydroxyl radical mediates this induction. Concordantly, GSH peroxidase overexpression in WI38 cells attenuated GADD153 mRNA induction by H₂O₂. However, GADD153 induction by arsenite was only modestly reduced in the same cells, suggesting a lesser contribution of peroxides to gene activation by arsenite. We also demonstrated that oxidative stress participates in the induction of GADD153 by UVC (254 nm) irradiation. Finally, both promoter-deletion analysis and point mutation of the AP-1 site in an otherwise intact promoter support a significant role for AP-1 in transcriptional activation of GADD153 by UVC or oxidant treatment. Indeed, exposure of cells to oxidants or UVC stimulated binding of Fos and Jun to the GADD153 AP-1 element. Together, these results demonstrate that both free-radical generation and thiol modification can transcriptionally activate GADD153, and that AP-1 is critical to oxidative regulation of this gene. This study further supports a role for the GADD153 gene product in the cellular response to oxidant injury.