High aerobic glycolysis is frequent in cancers. Glucose phosphorylation is under control of hexokinase which is found in the cytosol or bound to mitochondria. Glycolysis parameters (glucose, pyruvate and lactate) and hexokinase were evaluated in extracts of 15 human gliomas and of normal brain tissue. Extracts were run and analysed for glucose lactate and pyruvate content using a centrifugal automatic analyzer, Mitochondria fractions were separated from total extracts and hexokinase enzymatic activities were measured in both, using an original micro-method. Conditions of hexokinase assay were standardized in terms of substrate concentration and linearity. Mean hexokinase activity in gliomas was variable, 3 times lower than in normal tissues and mainly bound to mitochondria, although lactate/pyruvate ratios were found to be 3.5 to 5.4 times higher. Glycolytic profile of tumor tissues can be rapidly assayed and evalated glycolysis in tumors could constitute a basis for therapy using antiglycolytic strategies.