Hexachloro-1,3-butadiene (HCBD) is nephrotoxic in rats. Its toxicity is due to a multistep bioactivation pathway involving glutathione conjugation. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine resulting from further processing of the GSH conjugate of HCBD is oxidized in vitro and in vivo to the corresponding sulfoxide diastereomers by cytochromes P450 3A. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide diastereomers represent vinyl sulfoxides which are electrophiles. They are analogous to alpha,beta-unsaturated carbonyl compounds and may be conjugated with glutathione. This study presents experimental data for the different reactivity of the two diastereomers of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide with glutathione S-transferases in vitro. The structures of the individual diastereomers were assigned by stereoselective oxidation of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine with sodium periodate in the presence of chloroperoxidase. The two isolated diastereomers were incubated with rat liver and kidney cytosol in the presence of glutathione. In incubations with rat liver cytosol, the formation of a glutathione conjugate, which was identified as (R)-N-acetyl-S-(4-glutathion-S-yl-1,2,3,4-tetrachlorobutadienyl )-L-cysteine sulfoxide, was observed with the (R)-sulfoxide diastereomer. The enzymatic reaction of the (S)-sulfoxide diastereomer with glutathione resulted in two GSH conjugates identified as (S)-N-acetyl-S-(4-glutathion-S-yl-1,2,3,4-tetrachlorobutadienyl )-L-cysteine sulfoxide and (S)-N-acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl )-L-cysteine sulfoxide. In rat kidney cytosol only the S-diastereomer of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide is transformed to (S)-N-acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl )-L-cysteine sulfoxide, while transformation of the R-diastereomer to glutathione conjugates was not observed. In rat kidney cytosol, the rates of formation of (S)-N-acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl )-L-cysteine sulfoxide from conjugation of the S-diastereomer were comparable to those in rat liver cytosol. Incubation of (S)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide with purified rat and human glutathione S-transferases indicates that both R- and S-diastereomers were conjugated to the corresponding 1,4-disubstituted compounds by mu-glutathione S-transferases. Formation of the 1,2-disubstituted conjugation product of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide was catalyzed exclusively by alpha-glutathione S-transferases. These results are one of the first examples for differences in regio- and stereospecificity in reactions catalyzed by different glutathione S-transferase enzymes.