Abstract1,2,3,4-diepoxybutane (DEB), an in vivo metabolite of 1,3-butadiene (BD), is a carcinogen and mutagen. The strong carcinogenicity/mutagenicity of DEB has been attributed to its high DNA reactivity and cross-linking ability. Recently we have demonstrated that under in vitro physiological conditions (pH 7.4, 37 °C), the reaction of DEB with 2′-deoxyguanosine (dG) produced two diastereomeric pairs of the major nucleoside adducts resulting from alkylation at the N1-and N7-positions of dG, i.e., 2′-deoxy-1-(2-hydroxy-2-oxiranylethyl)-guanosine and 2′-deoxy-7-(2-hydroxy-2-oxiranylethyl)-guanosine, respectively [Zhang, X.-Y., and Elfarra, A. A. (2005) Chem. Res. Toxicol. 18, 1316]. As each of these adducts contains an oxirane ring, the abilities of these adducts to form cross-linking products with dG under physiological conditions were investigated. Incubation of the N7 nucleoside adducts and their corresponding guanine product with dG led to formation of 7,7′-(2,3-dihydroxy-1,4-butanediyl)bis[2-amino-1,7-dihydro-6H-purin-6-one] (bis-N7G-BD), a known DEB cross-linking product. Incubation of the N1 nucleoside adducts with dG led to formation of a pair of diastereomers of 2′-deoxy-1-[4-(2-amino-1,7-dihydro-6H-purin-6-on-7-yl)-2,3-dihydroxybutyl]-guanosine (N7G-N1dG-BD), which are novel cross-linking products. Interestingly, the reaction of DEB with dG in glacial acetic acid at 60 °C yielded different crosslinking products, which were characterized as 2-amino-9-hydroxymethyl-4-{4-[2-amino-9-or 7-(4-acetyloxy-2,3-dihydroxybutyl)-1,7-dihydro-6H-purin-6-on-7-or 9-yl]-2, 3-dihydroxybutyl}-8,9-dihydro-7H-[1,4]oxazepino[4,3,2-gh]purin-8-ol (PA2) and 9,9′-bis(4-acetyloxy-2,3-dihydroxybutyl)-7,7′-(2,3-dihydroxy-1,4-butanediyl)bis[2-amino-1,7-dihydro-6H-purin-6-one] (PA4). Collectively, these results increase our understanding of the chemical reactivity and crosslinking ability of DEB under both physiological and non-physiological conditions.