N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP), can arylate and oxidize protein and nonprotein thiols in the pathogenesis of APAP-induced hepatotoxicity. We report the first direct evidence for the formation of a labile ipso adduct between glutathione (GSH) and NAPQI using a combination of techniques including liquid chromatography/tandem mass spectrometry and liquid chromatography/NMR spectroscopy. Decomposition kinetics of the GSH-NAPQI ipso adduct and product ratios suggested that the ipso adduct was readily reversible back to NAPQI under neutral and basic conditions. The significance of the ipso adduct is that it may migrate from its site of formation to other cell compartments where it can either oxidize protein thiols or covalently modify them. Ipso adduct formation with protein thiols was demonstrated with a cysteine protease, papain, whose catalytic activity relies on the presence of an active site cysteinyl thiol. The formation and reactions of cysteinyl thiol ipso adducts of NAPQI provides significant new insights into possible reactions of quinone imines with cellular peptides and proteins.