One of the most characteristic phenotypes of rapidly growing cancer cells is their propensity to catabolize glucose at high rates. Type II hexokinase, which is expressed at high levels in such cells and bound to the outer mitochondrial membrane, has been implicated as a major player in this aberrant metabolism. Here we report the isolation and sequence of a 4.3-kilobase pair proximal promoter region of the Type II hexokinase gene from a rapidly growing, highly glycolytic hepatoma cell line (AS-30D). Analysis of the sequence enabled the identification of putative promoter elements, including a TATA box, a CAAT element, several Sp-1 sites, and response elements for glucose, insulin, cAMP, Ap-1, and a number of other factors. Transfection experiments with AS-30D cells showed that promoter activity was enhanced 3.4-, 3.3-, 2.4-, 2.1-, and 1.3-fold, respectively, by glucose, phorbol 12-myristate 13-acetate (a phorbol ester), insulin, cAMP, and glucagon. In transfected hepatocytes, these same agents produced little or no effect. The results emphasize normal versus tumor cell differences in the regulation of Type II hexokinase and indicate that transcription of the Type II tumor gene may occur independent of metabolic state, thus, providing the cancer cell with a selective advantage over its cell of origin.