The effects of nuclear-localized oxidative stress on both nuclear antioxidant systems, and the processes that they regulate, are not clearly understood. Here, we targeted a hydrogen peroxide (H2O2)-producing enzyme, D-amino acid oxidase ( DAAO), to the nucleus (NLS-DAAO) and used this to generate H2O2 in the nuclei of cells. On addition of N-acetyl-D-alanine ( NADA), a substrate of DAAO, to NLS-DAAO-transfected HeLa cells, a twofold increase in ROS production relative to untreated, transfected control was observed. Staining of cellular thiols confirmed that NLS-DAAO-induced ROS selectively modified the nuclear thiol pool, whereas the cytoplasmic pool remained unchanged. Furthermore, NLS-DAAO/NADA-induced ROS caused significant oxidation of the nuclear GSH pool, as measured by nuclear protein S-glutathionylation (Pr-SSG), but under the same conditions, nuclear Trx1 redox state was not altered significantly. NF-kappa B reporter activity was diminished by NLS-DAAO/NADA-stimulated nuclear oxidation. We conclude that nuclear GSH is more susceptible to localized oxidation than is nuclear Trx1. Furthermore, the attenuation of NF-kappa B reporter activity in the absence of nuclear Trx1 oxidation suggests that critical nuclear redox proteins are subject to control by S-glutathionylation during oxidative stress in the nucleus.