The stereochemistry of the metabolism of vic-dihaloalkanes to alkenes has been studied. This glutathione-dependent biotransformation may occur by two mechanism. The first mechanism involves the nucleophilic attack of glutathione on the substrate resulting in S-(beta-haloalkyl)glutathione formation; subsequent attack of a second thiol on the sulfur atom of the conjugate yields glutathione disulfide, ethylene, and halide ion. Alternatively, glutathione may abstract a halide ion from the substrate and form ethylene, halide ion, and glutathione sulfenyl halide. These pathways were distinguished by determining the stereoisomeric alkenes formed in the metabolism of meso- and racemic 2,3-dibromobutane, erythro- and threo-2-bromo-3-chlorobutane, and meso-1,2-dideutero-1,2-dichloroethane. The stereochemical configurations of the 2-butenes and 1,2-dideuteroethylene were determined by gas chromatography and by Fourier-transform infrared spectroscopy, respectively. When incubated with glutathione and rat liver cytosol, meso- and racemic 2,3-dibromogutane were converted exclusively to (E)- and (Z)-2-butene, respectively. On the other hand, erytho- and threo-2-bromo-3-chlorobutane were converted to a mixture of (E)- and (Z)-2-butene. meso-1,2-Dideutero-1,2-dichloroethane was converted exclusively to (Z)-1,2-dideuteroethylene. These results suggest that the 2,3-dibromobutanes are metabolized to 2-butenes by a direct E2 elimination, whereas 2-bromo-3-chlorobutanes undergo metabolism to 2-butenes by both an E2 elimination and a substitution-elimination sequence. However, 1,2-dihaloethane metabolism to ethylene proceeds only by the substitution-elimination mechanism; this result is consisent with the formation of ethylene-S-glutathionylepisulfonium ion, a possible reactive species involved in 1,2-dihaloethane mutagenicity.