Promotion of lung tumors in mice by the food additive butylated hydroxytoluene (BHT) is mediated by electrophilic metabolites produced in the target organ. Identifying the proteins alkylated by these quinone methides (QMs) is a necessary step in understanding the underlying mechanisms. Covalent adducts of the antioxidant enzymes peroxiredoxin 6 and Cu,Zn superoxide dismutase were detected previously in lung cytosols from BALB/c mice injected with BHT, and complimentary in vitro studies demonstrated that QM alkylation causes inactivation and enhances oxidative stress. In the present work, adducts of another protective enzyme, carbonyl reductase (CBR), were detected by Western blotting and mass spectrometry in mitochondria from lungs of mice one day after a single injection of BHT and throughout a 28-day period of weekly injections required to achieve tumor promotion. BHT treatment was accompanied by the accumulation of protein carbonyls in lung cytosol from sustained oxidative stress. Studies in vitro demonstrated that CBR activity in lung homogenates was susceptible to concentration- and time-dependent inhibition by QMs. Recombinant CBR underwent irreversible inhibition during QM exposure, and mass spectrometry was utilized to identify alkylation sites at Cys 51, Lys 17, Lys 189, Lys 201, His 28, and His 204. Except for Lys 17, all of these adducts were eliminated as a cause of enzyme inhibition either by chemical modification (cysteine) or site-directed mutagenesis (lysines and histidines). The data demonstrated that Lys 17 is the critical alkylation target, consistent with the role of this basic residue in NADPH binding. These data support the possibility that CBR inhibition occurs in BHT-treated mice, thereby compromising one pathway for inactivating lipid peroxidation products, particularly 4-oxo-2-nonenal. These data, in concert with previous evidence for the inactivation of antioxidant enzymes, provide a molecular basis to explain lung inflammation leading to tumor promotion in this two-stage model for pulmonary carcinogenesis.