Oxidative stress-responsive transcription is regulated in part through cis-active sequences known as antioxidant response elements (ARE). Activation through the ARE involves members of the CNC-subfamily of basic leucine zipper proteins including Nrf1 and Nrf2. In particular, Nrf2 has been shown to coordinate induction of genes encoding antioxidant and phase 2 metabolizing enzymes in response to stimulation with electrophilic compounds and exposure to xenobiotics. Here we show that the 65-kDa isoform of the Nrf1 gene functions as a repressor of Nrf2. Transient expression of p65Nrf1 suppressed Nrf2-mediated activation of ARE-dependent reporter genes in cells. Induction of endogenous ARE-genes is blocked in Hepa1c1c7 cells stably expressing p65Nrf1 leading to increased cell death. Consistent with these findings, electrophilic activation of ARE-gene expression is augmented by loss of p65Nrf1 function in Nrf1(-/-) fibroblasts, and the protective effects of oxidative preconditioning and ARE-gene expression are blocked in Nrf1(-/-) cells stably expressing p65Nrf1. Gel shift experiments demonstrated that p65Nrf1 binds the antioxidant response element as a heterodimer with small-Maf protein. Immunoprecipitation studies demonstrated that p65Nrf1 competes with Nrf2 for interaction with small-Maf protein and binding to the antioxidant response element in vivo. Together, these results demonstrate that p65Nrf1 has the potential to play an important role in modulating the response to oxidative stress by functioning as a transdominant repressor of Nrf2-mediated activation of ARE-dependent gene transcription.