Liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESIMS/MS) was used to measure DNA adducts of the carcinogen 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) with a microbore C-18 reversed-phase column. Quantification of the isomeric adducts N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) and 5-(deoxyguanosin-N2-yl)-2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (dG-N2-MeIQx) was achieved using synthetic, isotopically labeled internal standards. The reaction of the N-acetoxy ester of 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) with calf thymus DNA (ct DNA) resulted in formation of these adducts in a ratio of 5:1 (dG-C8-MeIQx:dG-N2-MeIQx). The detection limit by LC/ESI-MS/MS in the selected reaction monitoring (SRM) mode ([MH+ → MH-116]+) (loss of deoxyribose) approached 500 fg (1 fmol) of adduct standard, and 1 adduct per 108 DNA bases using 100μg of DNA following solid-phase extraction. The SRM analysis of rat liver DNA 24 h after an oral dose of MeIQx (10 and 0.5 mg/kg) revealed the presence of isomeric dG-MeIQx adducts at levels of 3.07 ± 0.84 and 0.45 ± 0.27 adducts per 107 bases, respectively. LC/ESI-MS/MS product ion spectra were acquired on both adducts from the elevated dose of MeIQx for unambiguous adduct identification. The contribution of dG-N2-MeIQx to the total adducts in vivo was significantly more important than that observed in vitro. dG-C8-MeIQx was the principal adduct formed at the 10 mg/kg dose, (dG-C8-MeIQx:dG-N2-MeIQx (3:2)); however, dG-N2-MeIQx was the major lesion detected at the 0.5 mg/kg dose (dG-C8-MeIQx:dG-N2-MeIQx 1:10). The striking differences between the relative amounts of dG-C8-MeIQx and dG-N2-MeIQx formed in vivo as a function of dose suggest that reactive esters of HONH-MeIQx other than N-acetoxy-MeIQx may be formed in vivo and react preferentially with the N2 atom of guanine, or that dG-C8-eIQx is removed at a significantly more rapid rate than dG-N2-MeIQx. The dG-N2-MeIQx adduct, previously thought to be a minor adduct, is likely to be an important contributor to the genotoxic damage of MeIQx.