S-(2-Hydroxy-3,4-epoxybutyl)glutathione (DEB-GSH conjugate) is formed from the reaction of 1,2:3,4-diepoxybutane (DEB) with glutathione (GSH), and the conjugate is considerably more mutagenic than several other butadiene-derived epoxides-including DEB-in Salmonella typhimurium TA1535 [Cho, S.-H., (2010) Chem. Res. Toxicol. 23, 1544-1546]. We previously identified six DNA adducts in the reaction of the DEB-GSH conjugate with nucleosides and calf thymus DNA and two DNA adducts in livers of mice and rats treated with DEB [Cho, S.-H. and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 706-712]. To define the role of GSH conjugation in 1,3-butadiene (BD) metabolism and characterize the mechanism of GSH transferase (GST)-enhanced mutagenicity of DEB, mutation spectra of BD and its metabolites in the absence and presence of GST/GSH and mouse liver microsomes were compared in the rpoB gene of Escherichia coli TRG8. The presence of GST considerably enhanced mutations. The mutation spectra derived from the DEB-GSH conjugate, the DEB/GST/GSH system, and the BD/mouse liver microsomes/GST/GSH system matched each other and were different from those derived from the other systems devoid of GSH. The major adducts in E. coli TRG8 cells treated with the DEB/GST/GSH system, the BD/mouse liver microsomes/GST/GSH system, or the DEB-GSH conjugate were S-[4-(N(7)-guanyl)-2,3-dihydroxybutyl]GSH, S-[4-(N(3)-adenyl)-2,3-dihydroxybutyl]GSH, and S-[4-(N(6)-deoxyadenosinyl)-2,3-dihydroxybutyl]GSH, indicating the presence of the GSH-containing DNA adducts in the systems. These results, along with the strong enhancement of mutagenicity by GST in this system, indicate the relevance of these GSH-containing DNA adducts.