The cytoplasmic glutathione S-transferase activity of rat liver has been shown to increase to 300–400% of control values after treatment of the animals with trans-stilbene oxide and this phenomenon has been further characterized in the present study. Quantitative immunological determinations showed that the content of glutathione S-transferase A, B and C together constituted 4.5% of the soluble proteins in the hepatic cytoplasm of untreated rats. The content rose to 12.9 and 17.4% after treatment with trans-stilbene oxide or a combination of trans-stilbene oxide, 3-methylcholanthrene and phenobarbital, respectively. It was demonstrated that the cytosolic fraction from induced liver contains 4.2 times as much antigen which can be precipitated with antiglutathione S-transferase B antiserum as does control cytosol. Antiglutathione S-transferase C, which interacts with transferases A and C, precipitates 3.3 times as much protein from the induced cytosol compared with control. Crossed immunoelectrophoresis and purification demonstrated that both A and C are increased in amount after treatment with trans-stilbene oxide. Thus, cytosolic glutathione S-transferases A, B, and C in liver are all induced by treatment of rats with trans-stilbene oxide. Immunological crossreaction, similar behavior during chromatography on CM-cellulose and hydroxypatite and similar specific activities suggest that the control and induced enzymes are essentially identical. trans-Stilbene oxide was found to serve as a relatively poor second substrate for glutathione S-transferases A, B and C and can thus be said to cause substrate induction of these enzymes.