Hydrogen peroxide (H2O2) at moderate steady-state concentrations synergizes with TNF-α, leading to increased nuclear levels of NF-κB p65 subunit and to a cell-type specific up-regulation of a limited number of NF-κB-dependent genes. Here, we address how H2O2 achieves this molecular specificity. HeLa and MCF-7 cells were exposed to steady-state H2O2 and/or TNF-α and levels of c-Rel, p65, IκB-α, IκB-β and IκB-ε were determined. For an extracellular concentration of 25 µM H2O2, the intracellular H2O2 concentration is 3.7 µM and 12.5 µM for respectively HeLa and MCF-7 cells. The higher cytosolic H2O2 concentration present in MCF-7 cells may be a contributing factor for the higher activation of NF-κB caused by H2O2 in this cell line, when compared to HeLa cells. In both cells lines, H2O2 precludes the recovery of TNF-α-dependent IκB-α degradation, which may explain the observed synergism between H2O2 and TNF-α concerning p65 nuclear translocation. In MCF-7 cells, H2O2, in the presence of TNF-α, tripled the induction of c-Rel triggered either by TNF-α or H2O2. Conversely, in HeLa cells, H2O2 had a small antagonistic effect on TNF-α-induced c-Rel nuclear levels, concomitantly with a 50 % induction of IκB-ε, the preferential inhibitor protein of c-Rel dimers. The 6-fold higher c-Rel/IκB-ε ratio found in MCF-7 cells when compared with HeLa cells, may be a contributing factor for the cell-type dependent modulation of c-Rel by H2O2. Our results suggest that H2O2 might have an important cell-type specific role in the regulation of c-Rel-dependent processes, e.g. cancer or wound healing.