The substrate specificity of purified rat liver glutathione S-transferases (GSTs) for a series of gamma-glutamyl-modified GSH analogues was investigated. GST isoenzyme 3-3 catalysed the conjugation of 1-chloro-2,4-dinitrobenzene with six out of the nine analogues. alpha-L-Glu-L-Cys-Gly and alpha-D-Glu-L-Cys-Gly showed catalytic efficiencies of 40% and 130% that of GSH respectively. The GSH analogue with an alpha-D-glutamyl moiety appeared to be a highly isoenzyme-3-3-specific co-substrate: kcat./Km with GST isoenzyme 4-4 was only about 5% that with GST isoenzyme 3-3, and no enzymic activity was detectable with GST isoenzymes 1-1 and 2-2. GST isoenzyme 4-4 showed some resemblance to GST 3-3: five out of nine co-substrate analogues were accepted by this second isoenzyme of the Mu multigene family. Isoenzymes 1-1 and 2-2, of the Alpha multigene family, accepted only two alternative co-substrates, which indicates that their GSH-binding site is much more specific.