The stereoselectivity of cytosolic glutathione S-transferases (GS-T) in rat tissues was determined using (+/-)-benzo(a)pyrene 4,5-oxide (BPO), (+/-)-benz(a)anthracene 5,6-oxide (BAO), pyrene 4,5-oxide (PO), and (+/-)-styrene 7,8-oxide (SO) as substrates. An HPLC system is described which separates the four diastereomeric glutathione (GSH) adducts of BPO. Liver, lung, testis, and heart cytosol were found to be highly selective for catalysis of the reaction of the GSH sulfur atom with R-configured oxirane carbon atoms of BPO; heart was the most stereoselective of these tissues with 93% of the products arising from thiol attack at the R-configured carbons. These same tissues showed identical but lower stereoselectivity with PO or BAO as substrate. With SO as substrate, GSH attack was primarily at the benzylic carbon atom of the R-configured enantiomer in all tissues. In contrast, kidney and spleen cytosol were highly stereoselective for reaction of GSH with S-configured oxirane carbon atoms of all three polycyclic arene oxides (R/S ratio = 0.2-0.3) and showed a greater amount of attack at the terminal carbon atom of (7R)-SO. Enantioselectivity of GS-T from these tissues with BPO as substrate varied substantially; liver, kidney, spleen, and intestine preferentially catalyzed reaction with (4R,5S)-BPO (2.8- to 5.1-fold), but testis, lung, and heart showed little or no enantioselectivity [(4R,5S)-BPO/(4S,5R)-BPO = 1.3, 1.3, and 0.96, respectively]. In general, the differences in stereoselectivity between different rat tissues correlate with known tissue differences in isozyme composition and demonstrate that some rat GS-T isozymes may have markedly different stereo- and enantioselectivities with chiral epoxide substrates.