6-Phosphogluconate activates phosphofructokinase in extracts of rat liver by decreasing both the apparent S0.5 for fructose 6-phosphate and the degree of cooperative binding of fructose 6-phosphate. There is no effect on the maximum velocity. Enzyme activity is a hyperbolic function of the concentration of 6-phosphogluconate, and the apparent Km is 60 microM. Thus, a substantial activation of the enzyme is achieved with a physiological concentration of 6-phosphogluconate (40 microM). Activation of phosphofructokinase by 6-phosphogluconate is influenced by a low molecular weight factor(s) in liver extracts. Part of the influence may be attributed to fructose 2,6-bisphosphate which, in some conditions, acts synergistically with 6-phosphogluconate to activate phosphofructokinase. In the presence of a mixture of ATP, ADP, and AMP at physiological concentrations, the effects of 6-phosphogluconate (40 and 200 microM) and a saturating concentration of fructose 2,6-bisphosphate (400 nM) are more nearly additive. This result suggests that 6-phosphogluconate and fructose 2,6-bisphosphate act at different sites on the enzyme and that 6-phosphogluconate may contribute to the physiological regulation of hepatic phosphofructokinase. Regulation of phosphofructokinase by 6-phosphogluconate may provide a means by which the disposition of glucose 6-phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis can be coordinated, an effect which may be important during hepatic lipogenesis.