The highly glycolytic hepatoma cell line H-91 is characterized by a high hexokinase activity relative to rat liver; 50% of this activity is associated with the mitochondrial fraction [Bustamante, E., & Pedersen, P. L. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3735-3739], Treatment of mitochondria from this cell line with adenosine 5'-triphosphate (ATP) or glucose 6-phosphate solubilizes bound hexokinase activity. Solubilization of the enzyme by ATP results in a six- to sevenfold purification. Free ATP, unchelated by Mg ions, induces the release of the enzyme from the membrane, whereas the MgATP complex is ineffective. Ethylenediaminetetraacetic acid (EDTA) fails to release mitochondrial hexokinase indicating that the enzyme is not attached to the membrane by divalent cations. Energization of mitochondria is not required for ATP to induce solubilization of bound hexokinase. This is evidenced by (a) the ability of the nonhydrolyzable ATP analogue adenylyl imidodiphosphate to solubilize the enzyme, (b) the inability of uncouplers and inhibitors of oxidative phosphorylation to either solubilize or prevent the release of mitochondrial hexokinase, and (c) the inability of atractyloside to solubilize or prevent the release of bound hexokinase. The bound and the ATP-solubilized forms of mitochondrial hexokinase from H-91 hepatoma cells are kinetically different. When membrane bound, the enzyme has a significantly higher apparent affinity (Km = 0.25 mM) for its substrate MgATP than when solubilized (Km = 1.2 mM). Free ATP acts as a competitive inhibitor of mitochondrial hexokinase. Both the membrane-bound and the solubilized forms of mitochondrial hexokinase have about the same apparent affinity for glucose (Km = 56 and 83 μM, respectively). The experiments reported here provide the first description of the properties and the nature of binding of mitochondrial hexokinase from a tumor cell line growing in tissue culture.