The outer mitochondrial membrane of beef heart was disrupted by hypotonic treatment and the effects of concentration and ionic strength of creatine kinase substrate solutions and some other electrolytes and non-electrolytes on mitochondrial membrane creatine kinase were studied. It was shown that electrostatic forces play an important role in the binding of creatine kinase to the mitochondrial membrane. It was assumed that under physiological conditions mitochondrial creatine kinase may undergo a reversible association - dissociation reaction with the membrane. The changes of pH within the range of 6.0 to 9.5 had no effect on the creatine kinase binding to mitochondrial membrane. Creatine kinase from beef heart mitochondria binds likewise and at the same rate to the rat liver no creatine kinase. The number of binding sites in mg of heart mitochondrial protein is n1 = 0.54 +/- 0.11 nmole, Kd1 = 0.16 +/- 0.04 microM. The number of binding sites in mg of liver mitochondrial protein is n2 = 0.65 +/- +/- 0.03 nmole, Kd2 = 0.29 +/- 0.09 microM. Excessive cytochrome c inhibits the binding of creatine kinase to the beef heart mitochondrial membrane. The results obtained suggest that mitochondrial creatine kinase is apparently bound to the membrane phospholipids.