1,3-Butadiene (BD) is a carcinogen in rats and mice. Previous in vitro studies showed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. Blood and tissue levels of BDE were significantly lower in rats than in mice following exposure to BD. Since mice are much more susceptible to cancer induced by BD than rats, these findings suggest a key role for BDE in BD-induced carcinogenicity. The aim of this study was to characterize the glutathione (GSH) conjugation of BDE by cytosol from human liver and mouse and rat liver and lung in vitro. BDE and radiolabeled GSH were incubated with cytosol. Conjugates were identified by 13C-NMR and FAB mass spectroscopy and quantitated by HPLC. The enzyme kinetics for the conjugation of BDE with GSH suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in GSH conjugation of BDE.