The minimum number of glutathione S-transferase species in adult chicken liver cytosol is four, as judged by O-(carboxymethyl)-cellulose chromatography. One species of enzyme, the second activity peak eluted from O-(carboxymethyl)-cellulose with a linear 0-200 mm KCl gradient, was purified by various procedures including DEAE-cellulose chromatography, ammonium sulfate precipitation, O-(carboxymethyl)-cellulose chromatography, and preparative isoelectric focusing. The purified protein had a specific activity of 3.4 units/mg of protein when 1-chloro-2,4-dinitrobenzene was used as a substrate. The molecular weight of the protein, as determined by gel filtration, was 45,000. Electrophoresis under denaturing conditions revealed a single band of molecular weight about 23,000, indicating the enzyme is composed of two subunits of approximately equal size. The enzyme had a pH optimum of 8.8, and isoelectric point of 8.9, and an apparent Km of 0.35 mm for reduced glutathione and 1.92 mM for the substrate 1-chloro-2,4-dinitrobenzene. The enzyme exhibited an obligate requirement for reduced glutathione as the thiol donor. Like the mammalian enzyme, the purified chicken hepatic glutathione S-transferase also demonstrated multiple substrate specificities and was able to conjugate 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, 1,2-epoxy-3-(p-nitrophenoxy) propane, trans-4-phenyl-3-buten-2-one, and ethacrynic acid. In addition to its catalytic activity, the purified transferase was also able to bind hemin. Fluorescence quenching studies of the binding of hemin to the enzyme indicated a dissociation constant of 2.2 × 10-8, m and a 1:1 molar ration of hemin binding to the purified enzyme. Kinetic analyses showed that hemin inhibited enzyme activity; inhibition by hemin was competitive with 1-chloro-2,4-dinitrobenzene but noncompetitive with reduced glutathione (Ki ∼- 10-6 m). The ability of the enzyme to bind hemin suggests a possible role for glutathione S-transferase in the modulation of free intracellular heme levels, which may regulate the induction of S-amino-levulinic acid synthase, the rate-limiting enzyme in the heme biosynthetic pathway. © 1980.