Many studies on the mechanism of the inotropic action of cardiac glycosides have shown the possible intimate relationship between the mobilization of intracellular calcium and inotropic effect. Evidence obtained from recent studies suggests that cardiac glycosides may increase the intracellular Ca⁺⁺ concentration through the release of this ion from cellular or intracellular membrane. It seemed imperative to study the effect of ouabain on the interaction between mitochondria and Ca⁺⁺, because mitochondria are known to have a rather powerful Ca⁺⁺ pump mechanism which may have an important role on the regulation of intracellular Ca⁺⁺ concentration. The present investigations was made into the effect of ouabain on Ca⁺⁺ untake of mitochondria in the presence of ATP and its dependence on K⁺ and Na⁺ in the medium. The results are summarized as follows: 1. The rate of rise in the turbidity of superprecipitation was solely influenced by ionic strength of the medium, not by the species of ion, i.e. Na⁺ or K⁺. The higher ionic strength suppressed and the lower enhanced the rate of superprecipitation respectively. 2. No effect of ouabain was found on the rate of superprecipitation. 3. Mitochondria depressed the rate of superpretipitation, and the depressed rate of superprecipitation by mitochondria was reversed by ouabain, and the degree of this reversal was almost identical in Na⁺ and K⁺ medium. 4. Ca⁺⁺ uptake of mitochondria was inhibited by ouabain in the presence of ATP and the degree of inhibition showed the dose-response manner in terms of concentration of ouabain. 5. In the absence of ATP, mitochondria took or the Ca⁺⁺ in initial period but released it later. Such uptake and release of Ca⁺⁺ was not influenced by ouabain. 6. It is suggested that intracellular calcium mobilization by ouabain through the action upon the mitochondria was due to inhibition on ATP-dependent Ca⁺⁺ uptake by this agent, not to the action upon so called binding.