Oxidative stress influences a variety of regulatory proteins, including nuclear factor-kappaB (NF-kappaB). NF-kappaB is critical for maintaining the proliferation/apoptosis balance in hepatocytes. In this study we investigated the causal links between glutathione, NF-kappaB and hepatocyte damage. HepG2 and 3B cells were exposed to different doses of H2O2 or N-acetylcysteine (NAC) and the proliferation/apoptosis rate, glutathione forms, and p65NF-kappaB glutathionylation and activity were analysed. Our results demonstrate that H2O2 stopped proliferative response at low doses, but induced apoptosis only at high doses. In contrast, NAC exerted, proportionally to its concentration, a dual role simultaneously increasing both proliferation and apoptosis. Interestingly, the levels of protein-bound glutathione were increased by H2O2 and decreased by NAC. Moreover, the antibody recognizing the glutathionylated proteins co-precipitated and -localized with the cytoplasmic inactive form of p65NF-kappaB in H2O2- and NAC-treated cells, even when, in 1 mM NAC-treated cells, a part of p65 was glutathione-free and localized into the nucleus. Apoptotic cells were characterised principally by a cytoskeletal staining of glutathionylation and retention of NF-kappaB in the cytoplasmic region; whereas in proliferating cells, glutathionylated proteins were concentrated into the perinuclear region and p65NF-kappaB was traslocated into the nucleus. While cytoplasmic NF-kappaB retention correlated well with an increased apoptotic rate, a greater expression of this protein was observed in association with the NAC-dependent. In conclusion, our findings suggest that glutathionylation inhibits NF-kappaB activity causing reduced hepatocyte survival, which is common in several liver diseases.