Two forms of pyruvate kinase, PK I and PK II, have been demonstrated in flounder liver. PK I, purified 991-fold to a specific activity of 105 units per mg of protein, has an unusually high molecular weight of about 2 X 10(6). PK II, purified 172-fold to a specific activity of 16.5 units per mg of protein, has a molecular weight of 210,000 when determined on a sucrose gradient but of 300,000 when derived from gel chromatography. PK I and PK II differ in sensitivity to the inhibitor L-phenylalanine, a fact which is used to evaluate the amount of each of them in a mixture. pH optimum for both forms is 6-6.6. PK I and PK II behave different in an Arrhenius plot--PK II showing a transition at 21 degrees C.