1,3-Butadiene (BD), a gas used widely in production and synthesis of rubber, plastics, and resins, is metabolized to three different epoxide metabolites: butadiene monoxide (BMO), butadiene bisoxide (BBO) and epoxybutanediol (ED). We have examined the role of stereochemistry in the chemical and enzyme-mediated conjugation of R- and S-BMO; RR-, SS-, and meso-BBO; and SR-, RS-, RR-, and SS-ED with glutathione (GSH) using liver cytosol and freshly isolated hepatocytes from male Sprague-Dawley rats. Chemical and enzyme-mediated reactivity of BD-epoxides (5 mM) with GSH (0.1 mM) was assessed by measuring remaining GSH in solution using dithiobisnitrobenzoic acid (DTNB). Chemical reactivity of BD epoxides with GSH was modest while addition of cytosol resulted in increased removal of GSH following exposure to each BD-epoxide. BBO stereoisomers resulted in the greatest cytosol-mediated removal of GSH (40-70%). BMO enantiomers removed 40-60% and ED stereoisomers removed 10-35% of GSH. Cytosol-mediated reactions with GSH were predictive of results observed in isolated hepatocytes where stereoselective depletion of GSH was observed following treatment with each class of epoxide metabolites. R-BMO depleted cellular GSH more rapidly and to a greater extent than S-BMO, SS- and meso-BBO were more potent than RR-BBO and SS- and SR-ED were more potent than RS- and RR-ED. These data demonstrate that enzyme-mediated reactions represent the primary mechanism of conjugation of BD-epoxides with GSH and that these reactions display marked stereoselectivity.