Two quinone methide (QM) metabolites of the phenolic antioxidant butylated hydroxytoluene (BHT), 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and the tert-butyl-hydroxylated derivative (BHTOH-QM), are believed to be responsible for promoting lung tumor formation in mice treated with BHT. QMs are strongly electrophilic and undergo Michael type additions with nucleophiles at the exocyclic methylene to form benzylic thioether adducts. Our goal was to identify intracellular protein targets of these QMs in order to gain insight into their effects on tumorigenesis. Cell line E10 of mouse lung epithelial origin and its spontaneous transformant, the tumorigenic E9 cell line, were treated with BHT-QM or BHTOH-QM, and cellular proteins were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Adducted proteins were detected on western blots with polyclonal antibodies developed to a conjugate of BHTOH-QM that recognized adducts of both QMs bound to thiol groups of Cys and side chain amino groups of Lys and His residues. Tryptic digests of immunoreactive proteins were analyzed by HPLC mass spectrometry (LC/MS) and identified by searching protein databases using MS/MS data. In a few cases, adducted peptides in these digests were detected by matrix-assisted laser desorption/ionization time-of-flight MS. A total of 37 immunoreactive proteins were identified including proteins involved in carbohydrate metabolism, nucleic acid synthesis, and RNA and protein processing, in addition to several cytoskeletal and stress-related proteins. About half of the protein adducts were found in both cell lines. Adducts detected only in transformed E9 cells include glutathione S-transferase P1, peroxiredoxin 2, nucleoside diphosphate kinase, and vinculin, whereas several alkylated cytoskeletal proteins such as tubulins, vimentin, calvasculin, and calcyclin were detected exclusively in E10 cells. Several of the proteins modified by BHT-derived QMs have been implicated in various aspects of tumorigenesis and are excellent candidates for further study into the consequences of alkylation on cellular transformation.