It has been shown for the first time that the outer mitochondrial membrane has a low permeability for ADP and can control its diffusion into cells in vivo. Respiration of saponin-skinned cardiac and skeletal muscle fibers is maximally stimulated by millimolar concentrations of external ADP. The apparent Km values for ADP are equal to 297 +/- 35 and 334 +/- 54 microM, respectively. After complete extraction of myosin with 0.8 M KCl, which fully preserves the intact structure of the mitochondria, the apparent Km values for exogenously added ADP does not change. However, disruption of the outer mitochondrial membrane by osmotic shock (treatment with 40 mOsM KCl) causes a reduction of the apparent Km value down to 32.3 +/- 5.0 microM of ADP. The apparent Km for ADP in isolated heart mitochondria is 17.6 +/- 1.0 microM. It is concluded that there exists an intracellular factor in the cells in vivo which controls the outer mitochondrial membrane and notably decreases its permeability for ADP. After isolation of mitochondria this factor is lost. When mitochondrial creatine kinase is activated, weak intracellular fluxes of ADP passing through the outer mitochondrial membrane in the skinned fibers are amplified manifold due to the tight functional coupling between mitochondrial creatine kinase and the oxidative phosphorylation system. This coupling is considered to be the central mechanism in the control of cell respiration.