Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that is involved in numerous pathologies, in part through stimulation of the mitochondrial production of reactive oxygen species (ROS). Previous studies show that in addition to mitochondrial superoxide dismutase- and glutathione-dependent systems, mitochondria also contain thioredoxin-2 (Trx2), an antioxidant protein that can detoxify ROS. The purpose of this study was to determine whether Trx2 protects against oxidative damage triggered by TNF-alpha. After a 30-min treatment in HeLa cells, TNF-alpha (5-40 ng/ml) oxidized Trx2 but not cytoplasmic Trx1. Preferential, significant Trx2 oxidation occurred within 10 min of TNF-alpha treatment. Moreover, overexpression of Trx2, but not Trx1, decreased TNF-alpha-induced ROS generation, suggesting mitochondrial compartmentation of ROS production and subsequent specific detoxification by Trx2, not Trx1. Overexpression of Trx2 or the active-site mutant C93S Trx2 was used to evaluate their downstream effects following TNF-alpha stimulation. Results showed that nuclear translocation of NF-kappaB was inhibited with Trx2 overexpression but not with the dominant negative active-site mutant C93S Trx2. Moreover, when cotransfected with a NF-kappaB-luciferase reporter and then treated with TNF-alpha, NF-kappaB activity was significantly attenuated with Trx2 overexpression but not with C93S Trx2 expression. Trx2 overexpression, but not C93S Trx2, significantly inhibited TNF-alpha-induced apoptosis as measured by terminal dUTP nick-end labeling assay. These findings support the interpretation that mitochondrial-generated ROS is a principal component in TNF-alpha-induced effects and that Trx2 blocks TNF-alpha-induced ROS generation and downstream NF-kappaB activation and apoptosis.