Conversion of the toxic vinyl halide 1,1-dichloroethylene (DCE) to S-(2-S-glutathionyl-acetyl)glutathione (GSCH2COSG) involves sequential acylation and alkylation of two glutathione (GSH) molecules by the microsomal DCE metabolite ClCH2COCl. To examine its possible role in DCE biotransformation, we synthesized the putative intermediate S-(2-chloroacetyl)glutathione (ClCH2COSG). In aqueous buffer, ClCH2COSG did not hydrolyze to release GSH, but instead underwent a two-step rearrangement to yield a cyclic product. Product analyses by liquid secondary ion mass spectrometry and 1H-13C heteronuclear correlation nuclear magnetic resonance spectroscopy indicated that rearrangement involved initial transfer of the chloroacetyl moiety from the cysteinyl thiol to the gamma-glutamyl alpha-amine. The cysteinyl thiol then displaced chloride from the 2-chloroacetyl methylene carbon to yield the cyclic product. Incubation of 2 mM ClCH2COSG with 20 mM GSH yielded approximately 4.5-fold more cyclic product than GSCH2COSG. ClCH2COSG alkylated oxytocindithiol and N-acetyl-L-cysteine to yield S-[2-(alkylthio)acetyl]glutathione adducts analogous to GSCH2COSG. S-2-Chloroacetylation products were absent. In reacting with thiols by alkylation and in decomposing by rearrangement, ClCH2COSG displayed properties strikingly different from those of ClCH2COCl. Although much less reactive than its acyl halide precursor, ClCH2COSG may display greater selectivity in covalent modification of cellular targets in DCE intoxication.