We have previously observed that extracellular Mg2+ influences the phosphofructokinase (PFK) activity of intact Ehrlich Ascites tumour cells (EATC). In this study we have investigated the mechanism by which Mg2+ modulates this key glycolytic enzyme in EATC made permeable to the cation by either digitonin or dextran sulphate. Results showed that when Mg2+ is freely permeable to the cytosol, the in vivo PFK activity, calculated as FDP/G6P ratio, is not increased as it is in intact cells. We also observed that in permeabilized cells Mg2+ determines the increase of glucose 6 phosphate (G6P), fructose 1,6 bisphosphate (FDP) and lactate production. We hypothesize that extracellular Mg2+ regulates PFK and glycolysis in these neoplastic cells not by entering the cytosol but by a specific interaction with the plasma membrane.