An improved method is described for the purification of glyoxalase I from rabbit liver. The method involves homogenization, ammonium sulfate precipitation followed by choloroform/ethanol precipitation, affinity chromatography on Blue Dextran-Sepharose 4B and chromatography on Sephadex G-100. The enzyme is specifically eluted from the affinity column by S-hexylglutathione, a competitive inhibitor of the enzyme. This procedure offers a convenient method for obtaining electrophoretically pure glyoxalase I in high yields (60--70%).