Numerous environmental stimuli alter cell functions by the induction of intracellular reactive oxygen species, such as superoxide and hydrogen peroxide (H2O2). These redox alterations can change the activity of kinases and phosphatases responsible for controlling intracellular signal transduction cascades important in determining how cells react to their environment. One such well known pathway includes nuclear factor-kappaB (NFkappaB); however, the exact redox-sensitive factors important in controlling H2O2-mediated activation of NFkappaB remain unclear. In the present study, we have investigated how intracellular clearance of H2O2, using a recombinant adenovirus expressing glutathione peroxidase-1 (GPx-1), modulates NFkappaB activation following UV irradiation, tumor necrosis factor-alpha, or H2O2 treatment of MCF-7 cells. Findings from these studies demonstrate that GPx-1 overexpression can down-regulate NFkappaB DNA binding, and transcriptional activation of an NFkappaB-dependent luciferase reporter, to varying extents following these environmental stimuli. Studies using dominant negative adenoviral vectors expressing IKKalpha(KM) and IKKbeta(KA) suggest that GPx-1-mediated H2O2 clearance appears to preferentially inhibit the activity of IKKalpha, but not IKKbeta. These studies demonstrate for the first time that redox regulation of NFkappaB activation by intracellular H2O2 may be specific for a unique subunit in the IKK complex. Such findings suggest that IKK kinases or IKK phosphatases may have unique redox-regulated components. These studies have shed mechanistic insight into the potential application of redox-modulating gene therapies aimed at altering NFkappaB activation following environmental injury.