The 600 X g particulate fraction, obtained from the homogenates of human heart muscle, contained large quantities of an atypical creatine kinase (CK-Z). Creatine kinase Z migrated cathodically relative to CK-MM on agarose gel electrophoresis, and was not inhibited by antibodies directed against human CK-MM and CK-BB. Creatine kinase Z had an apparent Km for Mg-ADP and creatine phosphate of 0.04 mmol/l and 1.3 mmol/l, respectively. This enzyme existed in two molecular forms; one form of molecular weight 33 000-38 000 in the presence of a buffer containing Tris-HCl (0.05 mol/l), EDTA (0.001 mol/l), and 2-mercaptoethanol (0.010 mol/l), pH 8.0; and another form having a molecular weight of 62 000-68 000 in the presence of a buffer containing sodium phosphate (0.020 mol/l) and EDTA (0.001 mol/l), pH 8.0. Creatine kinase Z had biochemical properties which were different from those of the other soluble creatine kinase isoenzymes (MM, MB and BB), but similar to those reported for mitochondrial creatine kinases isolated from other animal tissues.