The structures of cysteine conjugates of 3,5-di-tert-butyl-4-hydroxytoluene (BHT) and the binding sites of BHT metabolites on microsomal protein were investigated by 13C nuclear magnetic resonance (13C-NMR) and gas-liquid chromatography/mass spectrometry. The cysteine conjugates of 2,6-di-tert-butyl-4-hydroxymethylphenol (BHT-alcohol) and 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (quinone methide), which are metabolites of BHT found in rat liver and specifically reacts with thiol compounds, were prepared as alcoholic aqueous solutions. The molecular structure of the cysteine conjugate of BHT-alcohol agreed completely with that of quinone methide in 13C-NMR spectra or mass spectra. These spectra of both conjugates further showed that the conjugates are due to thioether binding between the 4-methyl group of metabolites and the sulfhydryl group of cysteine. When [14C]BHT-bound microsomes prepared in vitro were enzymatically hydrolyzed with Pronase E, the major radioactive material that eluted with methanol from a column of Amberlite XAD-2 and gave a positive ninhydrin reaction was identified as a cysteine conjugate of BHT by comparing its Rf values on TLC and mass spectrum. On the basis of the results, it was apparent that the binding site of activated substituents of BHT on protein was mainly the sulfhydryl group of cysteine residue.