(1) M2-Pyruvated kinase (M2-PK) [EC 2. 7. 1. 40] was purified from a crude extract of Yoshida ascites hepatoma 130 cells by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 column chromatography. This purified preparation appeared homogeneous on ultra-centrifugation and electrophoresis, and its molecular weight was estimated to be approximately 2.16xlO55 by Sephadex G-200 column chromatography. (2) Anti-M2 serum was prepared in this laboratory from the blood of a chicken immunized with purified M2-PK. Comparative studies on L, M1, and M2-PK's using anti-M1 and anti-M2 sera suggested that these three types of PK's were immunologically distinguishable. (3) Comparative studies on the kinetic properties of the three types of PK suggested that these enzymes differed qualitatively: (a) A plot of M2-PK activity against PEP concentration was sigmoidal similarly to that of L-PK. However, the cooperative interaction of M2-PK with PEP was lower than that of L-PK since the Hill coefficient of M2-PK for PEP was about 1.5. (b) Another similarity between the kinetic properties of M2-PK and L-PK was that both were activated by various organic solvents, such as ethylene glycol and glycerol. The activity curves of both PK's with respect to PEP concentration changed to the usual Michaelis-Menten type in the presence of 25% (v/v) ethylene glycol with disappearance of sensitivity to FDP. In contrast, the activity of M1-PK was not appreciably influenced by any organic solvent examined. (c) M2-PK was inhibited by ATP, the apparent K1 being 2.5xlO-3M. This value was similar to that of M1-PK but much higher than that of L-PK. The inhibition of M2-PK was scarcely reversed by addition of FDP unlike that of L-PK. Addition of Mg2+ ion did not reverse the ATP inhibition of L-PK, but reversed that of M2-PK partially and that of M1-PK completely, (d) L-Alanine strongly inhibited both L-PK and M2-PK, the apparent K1's being 1×10-3 and 6×lO-4M, respectively. However, the mechanisms of inhibition of the two PK's seemed different, since the inhibition of L-PK was cooperative and completely reversed by addition of FDP, whereas that of M2-PK was not reversed by FDP. L-Phenylalanine also inhibited both L-PK and M2-PK. However, M2-PK was much more strongly inhibited than L-PK: The apparent Ki's of L-PK and M2-PK were 5xlO“8 and 5xlO˜4M, respectively. L-Trypto-phan also inhibited L-PK and M2-PK, but Mi-PK was not inhibited by 5mM L-trypto-phan. Other amino acids did not have appreciable effects on the PK's. (e) PCMB inhibited the three types of PK in the order: L>M2>M1. (f) Evidence was obtained suggesting that there may be two interconvertible forms of M2-PK, an FDP-sensitive form and an FDP-insensitive one. However, this interconversion does not seem to involve a change in molecular weight. The present results indicate that the three types of PK differ qualitatively or are different enzymes.