Seven peaks showing glutathione S-transferase (GST) activity toward 1-chloro-2, 4-dinitrobenzene (CDNB) were separated from rabbit liver preparations by CM-52 cellulose column chromatography (pH 6.7). These peaks were designated as R1, R2, Rx, Ry, R3, Rz, and R4 in order of elution from the column. Among these peaks, R3 was found to have notable GST activity, which was responsible for the enzymatic formation of S-methylglutathione from fenitrothion [O, O-dimethyl O-(3-methyl-4-nitrophenyl)phosphorothioate] in the presence of glutathione. The GST (s) in peak R3 gave a characteristic zymogram after agar-gel electrophoresis as compared with other CM-52 column fractions.