Abstract The cytosolic glutathione S-transferases of rat liver have been fractionated by chromatofocusing into 10 distinct fractions based on their reactivity with 2,4-dinitrochlorobenzene. All these fractions were capable of generating leukotriene C 4 (LTC 4) from leukotriene A 4 (LTA 4) to some extent. An inhibitor of leukotriene synthesis, U-60,257, inhibited the activity of these enzymes. The cytosolic glutathione S-transferases of rat basophil leukemia (RBL) cells have been similarly fractionated. U-60,257 inhibited the activity of some of these fractions but not that of others. None of the fractions of the enzyme from RBL cells formed LTC 4 from LTA 4. The microsomal glutathione S-transferase from rat liver also produced LTC 4 from LTA 4. It differs from the microsomal LTC synthetase of RBL cells in at least two respects: (1) The enzyme from RBL cells did not react with chromophoric substrates like dinitrochlorobenzene while the enzyme from liver did react. (2) Triton X-100 potentiated the activity of the enzyme from basophil leukemia cells and solubilized it, while it inhibited the activity of the leukotriene-synthesizing enzyme in the rat liver preparation. These results, along with a distinctly different inhibitor profile, indicate that LTC synthetase is a new and distinct glutathione S-transferase.