The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria in the presence of Mg2+ and ATP increases from a value of 0.4 mumol min-1 mg-1 to 6-7 mumol min-1 mg-1 upon incubation for 5-6 h at 38 degrees C. The increase in activity does not occur in particles that have been passed through a Sephadex column. The activation is prevented and partially reversed by ATP. This indicates that the increase in hydrolytic activity is due to abolition of the inhibitory action of the natural ATPase inhibitor protein of Pullman and Monroy [(1963) J. Biol. Chem. 238, 3762-3769]. At maximal activation approximately 50% of the inhibitor protein of the starting preparation remains in the particles as inferred from direct assay of inhibitor protein content and by its interaction with 125I-labeled antibodies directed against the inhibitor protein. The extent of the activation, which presumably is an index of the equilibrium between active and inactive enzymes, is strictly dependent on salts. The rate of the activation depends on the concentration of salts and is favored by alkaline pH. From results of experiments on the effect of temperature on the rate of activation of the ATPase, it was calculated that the activation energy, delta H not equal to and delta S not equal to of the process were 53.34 kJ/mol, 50.83 kJ/mol and -158.99 J mol-1 K-1, respectively. The data indicate that in its native inhibiting state, the interaction of the inhibitor protein with the enzyme involves electrostatic interactions. Also it is concluded that abolition of the inhibitory action of the protein on ATPase activity is not compulsorily linked to release of the protein into the water space.