In the present study we have used both enzyme assay with 1-chloro-2,4-dinitrobenzene as substrate and immunochemical quantitation to examine the distribution of microsomal glutathione transferase in different organelles, in different organs, and in different organisms. This enzyme was found to constitute 3% and 5%, respectively, of the total protein recovered in the microsomal and outer mitochondrial membrane fractions from rat liver. Microsomal glutathione transferase present in other subcellular fractions can be accounted for by contamination by the endoplasmic reticulum. In contrast to the situation with rat liver microsomes the glutathione transferase activities of microsomes from extrahepatic tissues of this same animal could not be activated by treatment with N-ethylmaleimide. Nonetheless, significant albeit low levels of a protein with the same molecular weight and immunochemical properties as the rat liver enzyme could be detected in microsomes from several extrahepatic tissues, notably the intestine, the adrenal, and the testis. Of those mammals for which fresh liver could be obtained, all demonstrated N-ethylmaleimide-activatable glutathione transferase activity in their liver microsomes. On the other hand, representatives for fish, birds, and amphibia did not demonstrate such activatable transferase activity in their liver microsomes. Toad was the only species that had a notable (twofold) sex difference in their level of hepatic microsomal glutathione transferase activity.