1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen on the basis of epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its carcinogenicity in laboratory rats and mice. BD is metabolically activated to epoxide intermediates that can react with nucleophilic sites of cellular biomolecules. Among these, 1,2,3,4-diepoxybutane (DEB) is considered the ultimate carcinogenic species of BD due to its potent genotoxicity and mutagenicity attributed to the ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. DEB mutagenesis studies suggest that adducts formed at adenine bases may be critically important, as DEB induces large numbers of A ⇁ T transversion mutations. We have recently identified two regioisomeric exocyclic DEB-dA adducts, 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3- diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) and 1,N 6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (1,N6-α-HMHP-dA) (Seneviratne et al. ((2010) Chem. Res. Toxicol. 23, 118 -133), which were detected in DEB-treated calf thymus DNA and in tissues of BD-exposed laboratory animals. In the present work, we describe a column switching HPLC-ESI+-MS/MS methodology for the quantitative analysis of 1,N6-HMHP-dA isomers in the DNA of laboratory mice exposed to BD by inhalation. On the basis of their exocyclic structure, which prevents normal Watson-Crick base pairing, these adducts could be responsible for mutations at the A:T base pairs observed following exposure to DEB.