Previous work has shown that butylated hydroxytoluene [2,6-di-tert-butyl-4-methylphenol (BHT)] undergoes pi-oxidation in liver microsomes to form the quinone methide 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (QM). This electrophilic species binds covalently to glutathione and protein thiols and is believed to initiate pulmonary toxicity in mice. In the present investigation, we identified another quinone methide metabolite of BHT, 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylene-2,5-cyclohexadienone (QM-OH), formed subsequent to the microsomal hydroxylation of BHT at a tert-butyl group. Mouse liver and lung microsomes generate the two quinone methides, and evidence was obtained that both metabolites also are formed in vivo. In contrast, rat microsomes produce QM almost exclusively, with only traces of QM-OH formed in liver and none in lung. Studies of the chemical reactivities of the two quinone methides with GSH demonstrated that QM-OH reacts about 6-fold faster than QM. Infrared spectra, 1H NMR spectra, and electrochemical measurements all support the proposal that the enhanced electrophilicity of QM-OH is due to intramolecular hydrogen bonding of the ring oxygen with the side-chain hydroxyl. The results provide evidence, therefore, that the previous metabolic scheme for bioactivation of BHT to a pulmonary toxin should be amended to include tert-butyl hydroxylation and subsequent pi-oxidation to the activated electrophile QM-OH. This scheme is consistent with published data concerning BHT-induced pulmonary toxicity and provides an explanation for the species specificity of this effect.