The method of separation of tumor cell mitochondria (Ehrlich ascites cell and rat hepa toma cell, AH 130) by treating tumor cells with proteinase, by which the mitochondria are retained in a biologically active state, has been reported. The mitochondria thus obtained proved to have the normal activities of oxidative phosphrylation and respiratory control. The respiratery control of these mitochondria was nearly the same as those of liver mitochondria from normal rat but rather low in ADP/O ratio than the later. The tumor cell mitochondria also showed nearly a normal potency in the swelling at phase I and the oxidation-reduction of pyridine nucleotide, but the swelling at phase II which is induced by adding uncoupers such as oleate and Ca++, was markedly low as compared with that of rat liver mitochondria.