Glutathione S-transferases from rat liver were partially purified by ion exchange chromatography. Active peaks, tentatively identified as containing the 1-2, 2-2, 3-3, 3-4, 4-4 and 5-5 isoenzymes were kept for study. The glutathione conjugates, S-hexyl-, S-benzyl- and S-(2,4-dinitrophenyl) L-glutathione were tested as inhibitors of the enzymes. The 1-2, 2-2, 3-3 and 3-4 fractions were inhibited to similar extents by these conjugates. For all enzymes the hexyl conjugate at 0.1 mM concentration was strongly inhibitory, the benzyl conjugate moderately so and the dinitrophenyl compound was only weakly inhibitory. In contrast, the epoxide conjugating activity in the 4-4 and 5-5 peak was barely affected by the substituted glutathiones at 0.1 mM concentrations. Studies on a purified ligandin (isoenzyme 1-2) from rat liver showed that further metabolism of the glutathione conjugates, to the corresponding cysteines or mercapturic acids, resulted in products with inhibitory properties approximately three orders of magnitude less potent than those of the parent S-substituted glutathiones.