There are at least three major mammalian isozymes of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40), designated K4, L4, and M4. Whereas parenchymal cells from adult rat liver contain only the type L isozyme, parenchymal cells isolated from fetal and regenerating liver were found to synthesize both the K4 and L4 isozymes. A small amount of K-M hybrid was seen in regenerating liver, but there were no detectable M-L or K-L hybrids. Thus, it appears that type L pyruvate kinase is not synthesized at the same time in the same liver cell with either of the other two isozymes. The intermediate electrophoretic bands seen with homogenates of whole fetal liver, and in some earlier work attributed to either hybrid isozymes or to the presence of M4, are contributed by nonparenchymal cells which, in the fetus, are largely hemopoietic. These additional bands of pyruvate kinase are electrophoretically and immunologically similar to the pyruvate kinase isozymes found in adult erythrocytes. The results reported here suggest a very rigorous control in the synthesis of K4 and L4 isozymes in parenchymal cells of both fetal and regenerating liver as opposed to developing neurons and glia, where the shift from synthesis of type K to type M subunits appears to occur gradually and results in the production of substantial amounts of hybrid isozymes.