The specific activity and stereoselectivity of cytosolic glutathione S-transferases (GST) from various rabbit tissues and isolated cells were determined as an initial step in characterizing GST isoenzymes in the rabbit. Of the five tissues examined, liver cytosol had the highest specific GST activity with the polycyclic arene oxides (+/-)-benzo[a]pyrene 4,5-oxide (BPO), pyrene 4,5-oxide (PO) and (+/-)-benz[a]-anthracene 5,6-oxide (BAO), and kidney cytosol had the second highest. Lung, intestine and testis had relatively low activities with all three substrates. With the alkene oxide (+/-)-styrene 7,8-oxide (SO), testicular cytosol had the highest GST activity while liver cytosol had only half the activity of the testis. Cytosolic GST from liver and kidney were highly stereoselective for reaction of glutathione with the S-configured oxirane carbon atoms of BPO, PO and BAO, and all tissues but the intestine were enantioselective for (4R,5S)-BPO and (5S,6R)-BAO. With SO, the liver, kidney and testis preferentially catalyzed the reaction of glutathione with the benzylic carbon atom of (7S)-SO. There was virtually no enantioselectivity in lung cytosol with SO but a preference for reaction with (7R)-SO was noted in the intestine. The stereoselectivities found in the intestine with each of the four substrates were markedly different from the other tissues. Cytosol from isolated hepatocytes showed almost identical patterns of stereoselectivity with BPO and PO to those of whole liver cytosol. Similarly, the stereoselectivity of cytosol prepared from alveolar type II cells isolated from rabbit lung was the same as that of whole lung cytosol with these substrates, whereas cytosol of alveolar macrophages differed substantially from lung cytosol in both cases. There were marked differences in stereoselectivity of cytosol from freshly isolated Clara cells with BPO versus PO as substrate. With BPO, Clara cells were very similar to whole lung cytosol, but with PO they were not. The data are consistent with the differential tissue and cellular distribution of multiple GST isoenzymes in the rabbit.