1.At neutral pH, the temperature-dependent release of glutamate-oxaloacetate aminotransferase, glutamate dehydrogenase, malate dehydrogenase and B-hydroxybutyrate dehydrogenase from mitochondria in an in vitro system, is prevented as a function of the concentration of ATP, oxidizable substrates and ADP. On the other hand, orthophosphate causes the release of mitochondrial enzymes (optimum a t pH 6.5). This effect of Pi is completely inhibited by ADP but not by ATP. UTP and UDP partially replace ADP for this activity. The inhibitory effect of ADP is insensitive to either substrate or rutamycin.2. I n absence of added phosphate and a t acid pH values (optimum at pH 6.0), ATP or oxidizable substrates cause marked release of latent mitochondrial enzymes. ATP shows magnesium requirement for this activity. Inhibitors of electron transport and phosphoryl transfer reactions completely prevent these effects of ATP and substrates. Inhibitors of mitochondrial alkali metal cation uptake are inactive, Rutamycin and dinitrophenol prevent effects of ATP in freshly prepared but not in aged mitochondria.3. Chromatographically pure phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, but not saturated phospholipids, prevent phosphate from stimulating leakage of latent enzymes from mitochondria.4. Release of mitochondrial enzymes effected by oxidizable substrates, ATP, ADP and orthophosphate, but not phospholipids, seems to be associated with energy-dependent mitochondrial swelling.5. It is concluded that the temperature-dependent release of latent enzymes from mitochondria may depend on structural transformations of the mitochondrial membrane controlled in part by the oxidative phosphorylation mechanism.The limited permeability of the mitochondrial membrane to small anions is one of the main reasons for the apparent unavailability of a number of intramitochondrial enzymes to exogenous substrates. This fact is partly responsible for the phenomenon known as enzyme latency [i]. Recent evidence has suggested that availability of cytoplasmic substrates to latent enzymes in mitochondria may be controlled by: (a) the passive translocation of carboxylic substrate anions associated with energy-dependent uptake of alkali metal cations Permeability properties of mitochondrial membranes responsible for keeping large enzyme molecules within the particle are also important contributors Enzymes.