Nuclear factor kappa B (NF-κB) is a potent and pleiotropic transcription factor that can be activated by a wide variety of inducers, including interleukin-1 (IL-1). Although the detailed activation mechanism of NF-κB is still under investigation, it requires both phosphorylation and degradation of its inhibitory subunit IκB and the presence of an oxidative environment. In this study, we systematically evaluated the influence of glutathione peroxidase, glutathione reductase and catalase on IL-1-induced NF-κB activation by analysing the effect of specific inhibitors of these enzymes. For the three antioxidant enzymes mentioned, their inhibition correlated with an overactivation of NF-κB, particularly for glutathione peroxidase. Inversely, we tested the response of glutathione peroxidase-transfected cells on NF-κB activation, which was lower as compared with the parental cells. Furthermore, interleukin-6 production also correlated perfectly with the reduced level of NF-κB activation in these experiments. The results clearly show that NF-κB activation is, strongly dependent on the antioxidant potential of the cells, especially on the activity of reduced glutathione-dependent enzymes such as glutathione peroxidase. The results support the hypothesis that the level of the oxidised glutathione:reduced glutathione ratio and the activity of intracellular antioxidant enzymes play a major role in NF-κB fine tuning.