3,4-Epoxy-1-butene (EB) is the major mutagenic metabolite of butadiene (BD), an important industrial chemical classified as a probable human carcinogen. Although the mechanism of carcinogenicity of EB is not known, its reactions with nucleophilic sites of DNA giving pro-mutagenic lesions are likely to constitute the early crucial step in multistage carcinogenesis. This study was conducted to characterize the adducts formed from reactions of EB with the most nucleophilic DNA nucleobases, adenine (Ade) and guanine (Gua), as free nucleobases, 2'-deoxyribonucleosides and constituents of calf thymus DNA (CT DNA) in order to provide insight into the nature of DNA modification by EB. The adducts were isolated using HPLC separation coupled with diode array detection (DAD) and structurally characterized from their electronic, mass- and nuclear magnetic resonance spectra. Four EB-adenine products were identified as N-1-(2-hydroxyr3-buten-1-yl) adenine (EB-Ade I), N-1-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade II), N-3-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade III, and N-3-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade IV), Two previously reported guanine adducts: N-7-(2-hydroxy-3-buten-1-yl) guanine (EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-Gua II) were also collected. The purified adducts were used as reference compounds to detect and quantitate the corresponding adduct species formed in calf thymus DNA incubated with EB. All six adducts were detected in treated DNA, The N-7 position of guanine was the most reactive in DNA followed by N-3 of adenine and N-1 of adenine. The formation of N-1 and N-3-adenine adducts (EB-Ade I, 1.2 ± 0.36; EB-Ade II, 0.8 ± 0.27; EB-Ade III, 2.7 ± 0.38; EB-Ade IV, 5.9 ± 0.68 nmol/μmol Ade) in CT DNA was approximately one-tenth that of EB-guanine adducts (50.7 ± 2.37 and 47.9 ± 3.6 nmol/μmol Gua, respectively). The N-1-EB-Ade adducts detected in this study are likely to be the precursors of previously reported N6 - EB-adenine adducts through Dimroth rearrangement. Since BD and EB induce significant numbers of point mutations at A:T base pairs, the EB-adenine adducts may represent important lesions involved in BD-induced mutagenesis and carcinogenesis.