1,3-Butadiene (BD) is a major industrial chemical used in rubber and plastics production and is recognized as an animal and human carcinogen. Although the exact mechanism of BD-induced carcinogenesis is unknown, chemical reactions of epoxide metabolites of BD with DNA to form nucleobase adducts are likely to contribute to multistage carcinogenesis. Among BD-derived epoxy metabolites, 1,2:3,4-diepoxybutane (DEB) appears to be the most genotoxic and carcinogenic, probably because of its bifunctional nature. Initial DNA alkylation by DEB produces N7-(2′-hydroxy-3′,4′-epoxybut- 1′-yl)guanine monoadducts, which can then be hydrolyzed to N7-(2′,3′,4′-trihydroxy-1′-yl)guanine or can react with another site in double-stranded DNA to form 1,4-bis(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links. While (2′,3′,4′-trihydroxy-1′- yl)guanine lesions have been previously quantified in vivo, they cannot be used as a biomarker of DEB because the same lesions are also formed by another, more prevalent BD metabolite, 1,2-epoxy-3,4-butanediol. In contrast, bis-N7G-BD can only be formed from DEB, potentially providing a specific biomarker of DEB formation. We have developed a quantitative HPLC-ESI+-MS/ MS method for measuring racemic and meso forms of bis-N7G-BD in DNA extracted from tissues of BD-exposed laboratory animals. In our approach, bis-N7G-BD adducts are released from DNA as free bases by neutral thermal hydrolysis, purified by solid-phase extraction, and subjected to HPLC-ESI+-MS/MS analysis. Selected reaction monitoring is performed by following the loss of a guanine moiety from protonated molecules of bis-N7G-BD and the formation of protonated guanine under collision-induced dissociation. Quantitative analysis of racemic and meso forms of bis-N7G-BD is based on isotope dilution with the corresponding 15N-labeled internal standards. The lower limit of quantification of our current method is 10-20 fmol/0.1 mg of DNA. The accuracy and precision of the new method were determined by spiking control mouse liver DNA with racemic and meso forms of bis-N7G-BD (10 fmol each), followed by sample processing and HPLC-ESI+-MS/MS analysis. Calculated amounts of racemic and meso forms of bis-N7G-BD were within 20% of the theoretical value (9.7 ± 2 and 9.2 ± 1.9 fmol, respectively, N = 4). DNA extracted from liver and lung tissues of mice exposed to 625 ppm butadiene for 5 days contained 3.2 ± 0.4 and 1.8 ± 0.5 racemic adducts per 106 guanines, respectively, while the amounts of meso-bis-N7G-BD were below the detection limits of our method (1 per 107 guanines). Control animals did not contain either bis-N7G-BD lesion. Sensitive and specific quantitative methods for bis-N7G-BD analysis developed in this work provide a unique biomarker of DEB-induced DNA alkylation following exposure to BD.