A method was established for simultaneously isolating Theta-class glutathione (GSH) S-transferases (GSTs) T1-1 and T2-2 as homogeneous proteins from rat (r) liver cytosol. The established method of using an 8-aminooctyl Sepharose 4B column to separate rGSTT1-1 from rGSTT2-2 at the final stage of their purification was a modification of the method previously reported for the isolation of rGSTT2-2 (Hiratsuka et al., J. Biol. Chem., 265, 11973-11981, 1990). Specific substrates used for purification of the Theta-class rGSTs were dichloromethane for T1-1 and 5-sulfoxymethylchrysene for T2-2. rGSTsT1-1 and T2-2 existed at a ratio of 1:7 at a total concentration of 0.5% of that of the cytosolic protein. Purified rGSTsT1-1 and T2-2 were separated as single bands at 28 and 26.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as single peaks at retention times of 36 and 34 min, respectively, by reverse-phase partition high-performance liquid chromatography on a microBondasphere column eluted with a linear gradient of acetonitrile in water containing trifluoroacetic acid. Western blot analysis indicated that rabbit antisera raised against rGSTsT1-1 and T2-2 intensely reacted with the corresponding antigens, but showed no detectable reactivity with the different isoforms of Theta-class rGSTs as well as with representative hepatic rGSTs of other classes. The Theta-class rGSTs showed higher GSH peroxidase activity than rGSTA1-2 toward hydroperoxides of cumene, arachidonic acid, and linoleic acid. Cumene hydroperoxide was a better substrate for rGST T1-1 than for rGST T2-2, while the fatty acid hydroperoxides were the better substrates for rGST T2-2 than for rGST T1-1.