The following purification is presented: 0.051[image] tris buffer (pH 7.8) extraction of the acetone-dried powder of rat liver, heat treatment (52[degree]C, 5 min, pH 5.8), ammonium sulfate fractionation between 0.52 and 0.68 saturation, acid treatment (pH 4.3, 37[degree]C, 30 min.), alumina C gamma-gel column chromatography (the eluant: the increasing concentration of phosphate buffers), successively. The preparation is purified about 120 fold, stable between pH 5.8 and 7.2, fairly stable against acid treatment, and free of adenylate kinase, adenosine triphosphatase, nucleoside diphosphokinase or thiamine pyrrophosphatase.