1,2,3,4-Diepoxybutane (DEB), a metabolite of the carcinogen butadiene, has been shown to cause glutathione (GSH)-dependent base substitution mutations, especially A:T to G:C mutations in Salmonella typhimurium TA1535 [Cho, S. H., et al. (2010) Chem. Res. Toxicol. 23, 1544] and Escherichia coli TRG8 cells [Cho, S. H., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1522]. We previously identified S-[4-(N6-deoxyadenosinyl)-2,3-dihydroxybutyl]GSH [N6dA-(OH)2butyl-GSH] as a major adduct in the reaction of S-(2-hydroxy-3,4-epoxybutyl)glutathione (DEB-GSH conjugate) with nucleosides and calf thymus DNA and in vivo in livers of mice and rats treated with DEB [Cho, S. H., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 706]. For investigation of the miscoding potential of the major DEB-GSH conjugate-derived DNA adduct [N6dA-(OH)2butyl-GSH] and the effect of GSH conjugation on replication of DEB, extension studies were performed in duplex DNA substrates containing the site-specifically incorporated N6dA-(OH)2butyl-GSH adduct, N6-(2,3,4-trihydroxybutyl)deoxyadenosine adduct (N6dA-butanetriol), or unmodified deoxyadenosine (dA) by human DNA polymerases (Pol) η, ι, and κ, bacteriophage polymerase T7, and Sulfolobus solfataricus polymerase Dpo4. Although dTTP incorporation was the most preferred addition opposite the N6dA-(OH)2butyl-GSH adduct, N6dA-butanetriol adduct, or unmodified dA for all polymerases, the dCTP misincorporation frequency opposite N6dA-(OH)2butyl-GSH was significantly higher than that opposite the N6dA-butanetriol adduct or unmodified dA with Pol κ or Pol T7. LC–MS/MS analysis of full-length primer extension products confirmed that Pol κ or Pol T7 incorporated the incorrect base C opposite the N6dA-(OH)2butyl-GSH lesion. These results indicate the relevance of GSH-containing adducts for the A:T to G:C mutations produced by DEB.