Abstract Pyruvate kinase (ATP : pyruvate 2- O-phosphotransferase, EC 220.127.116.11) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R 2) is a heterotetramer designated as L 2L′ 2, the molecular weight of L′ being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R 1, predominant in the erythroblasts and the young red cells, is composed of four identical L′ subunits. 2. 2. A mild tryptic attack is able to transform PK-R 1 into PK-R 2, the PK-R 2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L′ subunits into L ones. 3. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L′ 4 enzyme secondarily partially proteolysed into L 2L′ 2. In liver a very active proteolytic system would be responsible for the total transformation into L 4 pyruvate kinase. 4. 4. L′ 4 enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L 4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S]0.5 of 1.2 mM. The characteristics of L 2L′ 2 are roughly intermediate between those of L′ 4 and of L 4. Fructose 1,6-biphosphate decreases [S] 0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphitenolpyruvate of these enzymes. 5.|Ls in4 pyruvate kinase is more inhibited by Mg. ATP than L′ 4 with L 2 L′ 2 the intermediate range. 6.|Tryptic trearment of each enzyme form studied transforms its kinetic behaviour into that observed for L 4.