The p53 tumor suppressor is found to be mutated and abundant in a wide variety of tumors. Within tumors showing rapid growth, the Type II isoform of hexokinase is also highly expressed to facilitate high rates of glucose catabolism, which in turn promote their rapid proliferation. We previously reported isolation of the proximal promoter of the Type II hexokinase gene from the highly glycolytic hepatoma AS-30D (Mathupala, S. P., Rempel, A., and Pedersen, P. L. (1995) J. Biol. Chem. 270, 16918-16925). Here, we show that a p53 protein, exhibiting two point mutations in its cDNA, is abundantly expressed in the AS-30D hepatoma. Co-expression studies showed that p53 overexpression significantly and reproducibly activated the Type II hexokinase promoter. Two functional p53 motifs were identified within this promoter by footprint and gel retardation analyses. Presence of functional p53 response elements on the Type II hexokinase promoter and the positive regulatory effect on the promoter by the mutant p53 indicates that in rapidly growing liver tumors, and perhaps in many other tumors as well, this highly abundant p53 protein plays a role in maintaining a high glycolytic rate. This is the first report of a possible link between loss of cell cycle control in rapidly growing cancer cells and their high glycolytic phenotype.