3,4-Epoxy-1-butene (EB) is generated by cytochrome P450-mediated epoxidation of 1,3-butadiene (BD), an important environmental and industrial chemical classified as a probable human carcinogen. The ability of EB to induce point mutations at GC and AT base pairs has been attributed to its reactions with DNA to form covalent nucleobase adducts. Guanine alkylation is preferred at the endocyclic N7 nitrogen, while adenine can be modified at the N1-, N3-, N7-, and the N6 positions. For each of these sites, a pair of regioisomeric 2-hydroxy-3-buten-1-yl and 1-hydroxy-3-buten-2-yl adducts is produced as a result of epoxide ring opening at the terminal C-4 or the internal C-3 carbon position of EB, respectively. The N6-EB-adenine adducts are of particular interest because of their stability in DNA, potentially leading to their accumulation in vivo. In the present work, synthetic DNA oligomers containing structurally defined N6-(2-hydroxy-3-buten-1-yl)-dA (N6-HB-dA) adducts were prepared for the first time by a postoligomerization approach that involved coupling 6-chloropurine-containing DNA with synthetic 1-amino-3-buten-2-ol. N6-HB-dA-containing DNA oligomers were isolated by reversed phase HPLC, and the presence of N6-HB-dA in their structure was confirmed by molecular weight determination from HPLC-ESI- -MS of the intact strands and by HPLC-ESI+-MS/MS and MS/MS/MS analyses of the enzymatic digests using synthetic N6-HB-dA as an authentic standard. N6-HB-dA-containing oligomers generated in this study will be used for structural and biological studies.