Rat liver 6-phosphofructokinase (ATP-D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was partially purified free of interfering enzymes by a rapid procedure. Fructose 2,6-bisphosphate, at micromolar concentrations, greatly stimulated the enzyme by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Its action was synergistic with that of AMP. As a stimulator of liver phosphofructokinase, fructose 2,6-bisphosphate was approximately 1000-and 2500-fold more efficient than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate, respectively. The concentration at which a half-maximal effect was obtained with the hexose bisphosphates was dependent upon the experimental conditions. It was relatively high at physiological concentrations of substrates, AMP, and Pi, and under these conditions the positive effect of fructose 1,6-bisphosphate was no longer detectable. This was probably due to the negative effect of fructose 1,6-bisphosphate as a reaction product inhibitor. It is concluded that fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate controls, in association with other effectors, the activity of phosphofructoldnase in the liver.There is a general agreement in the literature that 6-phosphofructokinase (ATP:D-fructose-6-phosphate l-phosphotransferase, EC 2.7.1.11) plays a major role in the control ofglycolysis in nearly all types of cells (1-6). The activity of this enzyme is controlled by several metabolites, most notably its two substrates, fructose 6-phosphate and ATP. The rate of the reaction is a sigmoidal function of fructose 6-phosphate concentration, and the saturation curve is shifted to the left in the presence of positive effectors, among which fructose 1,6-bisphosphate (7,8), the product of the reaction, and AMP are currently believed to play a major role. Negative effectors such as ATP and citrate have the opposite effect. The rate ofthe reaction plotted as a function of ATP concentration exhibits a typical inhibition by excess substrate. This inhibition is relieved by the positive effectors and by large concentrations of fructose 6-phosphate, whereas it is intensified by the negative effectors.Fructose 2,6-bisphosphate is a newly recognized, extremely effective, positive effector of liver and muscle phosphofructokinase (9). Its concentration in the liver is greatly increased under conditions in which glycolysis is active and is decreased by glucagon (10). It has also been found to inhibit, at micromolar concentrations, liver and muscle fructose-1,6-bisphosphatase (11). It therefore appears to be a major regulator of both glycolysis and gluconeogenesis in the liver. The purpose of the present work was to investigate the action of fructose 2,6-bisphosphate on the kinetics of liver phosphofructokinase and to compare it to that of its isomers fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and that of AMP. When the effect of fructose 1,6-bisphosphate was examined, pyruvate kinase (20 ,g/ml) and lactate dehydrogenase (25 Mg/ ml) were u...