Menadione (vitamin K-3,2-methyl-1,4-naphthoquinone), a radox cycling reagent, generates reactive oxygen intermediates and causes oxidative injury. The addition of menadione to Hep G2 cells produced a time- and concentration- dependent loss of cell viability. Preincubation of Hep G2 cells with low, nontoxic concentrations of menadione increased the viability of the cells against toxic doses of menadione or H2O2. Maximum protection was found with menadione concentrations of ~3 μM and preincubation times of ~45 min. This protective effect could be blocked by the protein synthesis inhibitor cycloheximide and by a variety of antioxidants. The transcription factor nuclear factor-κF (NF-κB) is known to be activated by many compounds, including reactive oxygen intermediates. Menadione activated NF-κB as determined by electrophoretic mobility shift assays. This activation was prevented by the same antioxidants that blocked protection against cytotoxicity produced by preincubation with menadione. Anti-p50 IgG prevented the menadione-stimulated binding of NF-κB to the oligonucleotide probe, whereas anti-p65 IgG produced a supershift of the NF-κB/oligonucleotide complex. Salicylate prevented the activation of NF-κB by menadione, and under these conditions, salicylate potentiated the cytotoxicity of menadione or H2O2. Transfection with a plasmid containing cDNA encoding mouse IκBβ, an inhibitor of NF-κB, resulted in increased toxicity by menadione. Furthermore, when protein kinase C was down-regulated by prolonged treatment with active phorbol ester (phorbol-12-myristate-13-acetate), the Hep G2 cells became more sensitive to menadione treatment. However, short term treatment with PMA, which activated NF-κB, resulted in protection against menadione cytotoxicity. Menadione cytotoxicity was enhanced when the Hep G2 cells were depleted of GSH. An increased level of GSH was observed after menadione pretreatment; this increase was blocked by salicylate, thereby linking the GSH increase to activation of NF-κB by menadione. The results of the current study suggest that menadione pretreatment protects Hep G2 cells from oxidative injury through an NF-κB-related mechanism, which may involve, in part, increased production of GSH.