Three of the isozymes of glutathione S-transferase (EC 2.5.1.18) from rat liver (isozymes A, B, and C) catalyze the addition of glutathione to phenanthrene 9, 10-oxide with varying degrees of efficiency and stereoselectivity. Isozyme C is 2-fold and 35-fold more efficient toward this substrate than are isozymes A and B, respectively, and gives a 20 to 1 ratio of the two possible diastereomeric products. The stereoselectivities of isozymes A (approximately 1 to 1) and B (3 to 1) are considerably lower. The major product diastereomer from isozyme C is deduced to have the 9S, 10S absolute configuration by circular dichroism spectroscopy, implying attack of glutathione on the oxirane carbon on R absolute configuration. Isozyme C shows little kinetic discrimination between other K-region arene oxides such as pyrene 4,5-oxide and the enantiomers of benz[a]anthracene 5,6-oxide and benzo[a]pyrene 4,5-oxide. However, the stereoselectivity toward all the substrates is conserved with predominant (greater than 95%) attack at the oxirane carbon of R absolute configuration to give the S,S product. The stereoselectivity of isozyme C is very sensitive to the introduction and location of nitrogen substitution in the phenyl rings of phenanthrene 9,10-oxide. As a result isozyme C shows little or no stereoselectivity toward 4,5-diaza- and 4-azaphenanthrene 9,10-oxide. In contrast, 1-azaphenanthrene 9,10-oxide is attacked preferentially at the R carbon of the oxirane. The results suggest that hydrophobic interactions between the enzyme surface and the substrate distal to the oxirane ring are important in determining the stereoselectivity of the enzyme toward arene oxides.