The carcinogen ethylene dibromide (EDB) is bioactivated via a pathway involving initial conjugation with the tripeptide glutathione (GSH) in a reaction catalyzed by GSH S-transferase. The conjugate then reacts preferentially with DNA guanyl residues to generate S-[2-(N7-guanyljethyl]glutathione. Rates of hydrolysis and alkylation of 4-(p-nitrobenzyl)pyridine with several cysteinyl and homocysteinyl analogues of 5-(2-haloethyl)glutathione at pHs 2.2, 6.4, and 8.5 are consistent with the hypothesis that an episulfonium ion is a common intermediate in both the hydrolysis and alkylation reactions. Consistently, 2-amino-6-chlorohexanoic acid failed to react with 4-(p-nitrobenzyl)pyridine. The stereochemical course of the overall reaction was studied with [threo-1,2-2M2]EDB and [erythro-1,2-2H2]EDB, which were incubated with GSH, rat liver cytosol, and DNA; the resulting DNA N7-guanyl adducts were isolated and analyzed by NMR techniques in order to determine the stereochemical course of the reaction. Two-dimensional correlated (COSY) NMR indicated that the reaction had occurred by a single stereochemical course. The magnitude of nuclear Overhauser effects between the ethylene protons suggests that the reaction occurs with net inversion of configuration of the methylene protons. This conclusion was confirmed upon comparison of the COSY NMR spectra of the biologically generated adducts with those that were synthetically prepared from the deuteriated EDB diastereomers via a known stereochemical route. This observation, combined with the kinetic data, supports a reaction mechanism where the EDB-GSH conjugate forms an episulfonium ion prior to reaction with DNA guanyl residues.