Rat liver cytoplasm (postnuclear supernatant) has a low aerobic glycolytic rate in the presence of added glucose, ATP, ADP, Pi, and NAD+, whereas cytoplasm from Ehrlich ascites tumor cells exhibit a high aerobic glycolytic rate which is typical of rapidly proliferating tumor cells. Tumor mitochondria, unlike liver mitochondria, contain bound hexokinase which constitutes about 70% of the total cellular hexokinase activity. The high aerobic glycolytic rate of Ehrlich tumor cytoplasm is reduced markedly if the mitochondria are removed and can be restored almost completely upon addition of the hexokinase-containing tumor mitochondria to tumor cytosol (postmitochondrial supernatant). Addition of tumor mitochondria to liver cytosol can enhance its glycolytic rate to levels approaching those of tumor cytoplasm, whereas added liver mitochondria are without effect on the already low glycolytic rate of liver cytosol. Addition of tumor mitochondria to tumor cytosol increases its glycolytic rate to the level of tumor cytoplasm, as mentioned above, but liver mitochondria added to tumor cytosol actually depress its glycolytic rate to the level of liver cytosol. The stimulatory effect of tumor mitochondria on liver cytosol can be ascribed to its associated hexokinase activity since hexokinase specifically removed from mitochondria of tumor cells can also enhance the glycolytic rate of liver cytosol. The depressing effect of added liver mitochondria on tumor cytosol glycolysis suggests that liver mitochondria can compete more effectively than tumor mitochondria for a common intermediate and/or cofactor. Examination of 12 different tumor cell lines revealed that only those which reached maximum size in 1 month or less, and which have elevated glycolytic activities, had detectable mitochondrially associated hexokinase activity. The studies reported here describe resolution and reconstitution of tumor cytoplasm, supplementation of cytosol with intact mitochondria or mitochondrial hexokinase, and a survey of mitochondrial hexokinase content in various tumors, and provide strong evidence for the view (Bustamante, E., and Pedersen, P. L. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3735-3739) that a form of hexokinase with a propensity for mitochondrial binding plays a key role in the high aerobic glycolysis of cancer cells.