2-tert-Butyl-1,4-benzoquinone (TBQ), an electrophilic metabolite of butylated hydroxyanisole (BHA), causes activation of Nrf2 together with S-arylation of its negative regulator Keap1 in RAW264.7 cells. In a previous study, we found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) covalently modified with 1,2-naphthoquinone (1,2-NQ) undergoes S-transarylation by GSH, resulting in a decline of the GAPDH-1,2-NQ adduct and formation of a 1,2-NQ-SG adduct ( Miura , T. et al. ( 2011 ) Chem. Res. Toxicol. 24 , 1836 -1844 ). In the present study, we explored the possibility of GSH-dependent S-transarylation of the Keap1-TBQ adduct. Pretreatment with l-buthionine-(S,R)-sulfoximine and N-acetylcysteine prior to TBQ exposure of HepG2 cells suggested that the Keap1-TBQ adduct appears to undergo GSH-mediated S-transarylation because the resulting alterations in the intracellular GSH concentration affected Nrf2 activation caused by TBQ. In support of this hypothesis, a cell-free study demonstrated that incubation of the Keap1-TBQ adduct with GSH results in the removal of TBQ from Keap1 with the production of mono- and di-GSH adducts of TB(H)Q. These results suggest that GSH plays a role in reversible covalent modification of TBQ derived from BHA to Keap1 through the formation of a C-S bond.