||An investigation of the physical properties of ATP-creatine transphosphorylase isoenzymes from rabbit, calf, and human has been made. The isoenzymes isolated from the same species differ widely in the net charge and is illustrated by liquid-boundary electrophoresis for the isoenzymes isolated from calf. Determinations of molecular weight of the kinetic unit of the isoenzymes were made by sedimentation equilibrium to provide a more quantitative comparison for the ultracentrifugal studies were made in the presence of dissociating and denaturing solvents and the values were found to 82,600; 87,000; 83,200; 85,600; and 88,400 for the enzymes isolated from rabbit muscle, rabbit brain, calf muscle, calf brain, human muscle, and human brain, respectively. The effects of several dissociating and denaturing agents to the enzyme isolated from rabbit muscle was carefully explored in order to establish the conditions for the study of the polypeptide chains of the isoenzymes which were isolated from rabbit, calf, and human. Physical measurements in dissociating and denaturing solvents showed that the enzyme molecules are composed of two non-covalently linked polypeptide chains with similar, if not identical, physical and chemical properties. The polypeptide chain of the enzyme from the cerebral tissue may be slightly heavier than the muscle counterpart.