Glutathione S-transferases in testis microsomes were purified from rats and compared with the liver microsomal transferase. When microsomal fractions were prepared from rat testis by the same method as used for liver microsomes, testis microsomal glutathione S-transferase activity was increased 2-fold by N-ethylmaleimide as compared to a 7-fold increase in that of the liver transferase. In contrast to the single glutathione S-transferase in liver microsomes, at least three isozymes of glutathione S-transferase were separated from testis microsomes on hydroxylapatite column chromatography. The major fraction exhibiting glutathione S-transferase activity from the testis microsomes was shown to contain a member of the Mu family. The second fraction with transferase activity contained one of the Alpha class, and the third and smallest fraction was found to contain the liver microsomal form of glutathione S-transferase. Since the GSH S-transferase of the Mu family is present in the cytosol, we isolated the GSH S-transferase from testis cytosol, it being suggested that the major GSH S-transferase in testis microsomes is the cytosolic transferase. These results indicate that testis microsomes contain mainly the cytosolic form of glutathione S-transferase, and that the activity of the liver microsomal form of the transferase is very low.