1. Purification of four isozymes of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) L, M1, M2 and R was much improved to give good yields by affinity elution chromatography. The enzyme was eluted from a phosphocellulose column with 0.5 mM phosphoenolpyruvate. Types L, M2 and R were stabilized with fructose 1,6-diphosphate throughout the purification procedures. 2. The isozymes were crystallized under various conditions: types L and R were readily crystallized from medium of low ionic strength, types L, M1, and M2 were crystallized from ammonium sulfate solution in different forms in the presence and absence of phosphoenolpyruvate. Type M1 was also crystallized in different forms in the presence and absence of fructose 1,6-diphosphate. 3. Amino acid analyses showed that the compositions of types L and R, and of types M1 and M2, respectively, were very similar.