1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. BD is metabolically activated by cytochrome P450 monooxygenases to 3,4-epoxy-1-butene (EB), which alkylates DNA to form a range of nucleobase adducts. Among these, the most abundant are the hydrolytically labile N7-guanine adducts such as N7-(2-hydroxy-3-buten-1-yl)-guanine (N7-EB-dG). We now report that N7-EB-dG can be converted to the corresponding ring open N6-(2-deoxy-d- erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine (EB-Fapy-dG) adducts. EB-Fapy-dG lesions were detected in EB-treated calf thymus DNA and in EB-treated mammalian cells using quantitative isotope dilution nanoLC-ESI+-MS/MS. EB-Fapy-dG adduct formation in EB-treated calf thymus DNA was concentration dependent and was greatly accelerated at an increased pH. EB-FAPy-dG adduct amounts were 2-fold higher in base excision repair-deficient NEIL1-/- mouse embryonic fibroblasts (MEF) as compared to isogenic controls (NEIL1+/+), suggesting that this lesion may be a substrate for NEIL1. Furthermore, NEIL1-/- cells were sensitized to EB treatment as compared to NEIL1+/+ fibroblasts. Overall, our results indicate that ring-opened EB-FAPy-dG adducts form under physiological conditions, prompting future studies to determine their contributions to genotoxicity and mutagenicity of BD.