Dog liver glutathione S-transferase activities are associated with five cytosolic proteins and to approximately 1.5% with microsomal proteins determined on the basis of activity conjugating to l-chloro-2,4-&nitrobenzene. The four major cytosolic enzymes were purified to apparent homogeneity by sequential use of ion-exchange, hydrophobic, hydroxyapatite and affinity chromatography. The isolated transferases are binary combinations of three classes of subunits: c1 (Mr = 26000), fi ( M , = 27000), y ( M , = 28500). They were classified by roman numerals assigned in order of increasing isoelectric point as DI,, (PI 6.4), DII, (PI 6.9), DIII,,, (PI 8.1), and DIV,, (PI 8.7). Additionally, traces of conjugating activity may be attributed to a, fi monomeric or dimeric protein with cationic character.The differences in catalytic specificity, temperature and pH dependence of activity, and sensitivity and kinetic response to inhibitory ligands may reflect the intrinsic structural heterogeneity of the transferases. At physiological glutathione concentrations D I?, accounted for roughly 60% of the total 1 -chloro-2,4-dinitrobenzene-conjugating activity, the rank order of activity being DI,, > D1Iua > DIV,, > DIII~,. The glutathione-dependent denitration of organic nitrates seems to be restricted to the cationic enzymes, whereas 1,2-dichloro-4-nitrobenzene-conjugating activity is exclusively associated with the anionic transferases, D I,, % DII,,. Arrhenius plots from initial rate experiments performed over a range of temperatures (15 -40°C) exhibit an upward bend for DI,,, an apparently constant slope for D II,, and D III,,, and a downward bend for D IVDy.