The distribution of the isozymes of creatine kinase [EC 2.7.3.2] of muscle and brain tissues on starch-gel electrophoresis was investigated by means of the direct extraction method. The intracellular distribution of creatine kinase in these tissues was also studied. The following results were obtained: 1) There were three isozymes of creatine kinase in the 20,000 ×g supernatant of human fetal skeletal muscle and two isozymes in that of adult rabbit muscle. Two isozymes of creatine kinase were present in the 20,000 x g supernatant of human fetal brain and two isozymes in that of adult rabbit brain, although one of the latter was very unstable and sometimes undetectable. 2) The intracellular distribution of creatine kinase activity was similar in adult rabbit muscle and brain tissues, that is, the cell sap as well as the microsomal fraction had markedly higher activity than the mitochondrial fraction. The nerve ending fraction in brain tissue also had an enzyme activity comparable with that of mitochondria. 3) The creatine kinase of cell sap had one or two isozymes showing similar electro-phoretic mobility to those of the microsomal fraction in the brain or muscle tissues of the adult rabbit, although the enzymatic activities of the two subcellular fractions were rather different in the two kinds of tissues. The major isozymes of cell sap and the microsomal fraction of brain tissue showed higher electrophoretic mobility than those of muscle tissue. 4) Rabbit muscle mitochondria had a specific type of isozyme migrating near the origin, while rabbit brain mitochondria had two kinds of creatine kinase which were of supernatant and mitochondrial types on starch-gel electrophoresis.