Reactive oxygen species (ROS) are used as signaling molecules in T cell activation. One of the main targets of ROS is the transcription factor nuclear factor-kappa B (NF-kappa B). NF-kappa B-dependent transcription is inhibited by antioxidants, and the activation is induced or potentiated by ROS. However, chronic oxidative stress is known to reduce the activation of T cells and NF-kappa B. To analyze these phenomena in more detail, we have exposed Jurkat T cells in vitro to oxidative stress (H2O2) at various times before or simultaneously with signals known to activate NF-kappa B (phorbol dibutyrate (PDBu) and TNF). Simultaneously applied H2O2 strongly potentiated the PDBu- or TNF-induced transcriptional activity of NF-kappa B. In contrast to this, H2O2 given 3 to 20 h before the activating signal reduced NF-kappa B-dependent transcriptional activity. This was not due to the oxidation-induced modification of NF-kappa B; cytoplasmic NF-kappa B was able to bind to DNA after dissociation from I kappa B alpha by detergent treatment. H2O2 pre-exposure effectively inhibited the PDBu- or TNF-induced phosphorylation and degradation of I kappa B alpha, but H2O2 given simultaneously with PDBu or TNF enhanced the degradation. Oxidative stress was also followed by a strongly decreased ability to form intracellular ROS. Taken together, these data indicate that I kappa B alpha phosphorylation is the target of action of ROS, and as the ROS-forming capacity is weaker after chronic oxidative stress, I kappa B alpha is not effectively phosphorylated and degraded, thus leading to decreased NF-kappa B-dependent transcription.