Benzoquinones (BQ) are genotoxic species that stem from metabolism of phenolic compounds. We have developed a method for measuring adducts of 1,2- and 1,4-benzoquinone (1,2-BQ and 1,4-BQ) with cysteine residues of hemoglobin (Hb) and albumin (Alb). The method employs a reductive catalyst (Raney® nickel) to selectively cleave sulfur-bound adducts so that they may be extracted with an organic solvent, derivatized with heptafluorobutyrylimidazole and measured by GC-ECD or GC-MS. Reactions of 1,4-BQ (0–300 μM) with whole blood of both F344 rats and humans resulted in linear formation of adducts with Hb and Alb. Adducts of 1,2-BQ with human Alb were formed by activation of catechol (0–300 μM) in situ with horseradish peroxidase. The mean half lives in vitro of 1,4-BQ in blood from humans and rats were 3.5 and 0.68 h, respectively. Second-order rate constants for reaction of 1,4-BQ with Hb and Alb in whole blood were estimated to be 18 and 76 1/mol/h for humans and 180 and 74 l/mol/h for rats respectively. Interestingly, the following high background levels of 1,2-BQ and 1,4-BQ adducts were observed in the proteins: 1,2-BQ-human Hb = 1.5 nmol/g, rat Hb = 25.1 nmol/g; human Alb = 8.0 nmol/g; 1,4-BQ-human Hb = 27.2 nmol/g, rat Hb = 11.5 nmol/g; human Alb = 20.5 nmol/g. These large background levels of BQ adducts suggest that significant exposure to BQ precursors occurs from dietary and/or endogenous sources. Given current evidence regarding the genotoxicity of BQ, such exposures and their health consequences should be investigated.