Addition of serum to quiescent cultures of 3T3 cells markedly enhances the specific activity of phosphofructokinase assayed in centrifuged homogenates. The effect depends on both serum concentration and the time of exposure to serum. Mixing experiments, mearsurements of the enzyme at different pH values and persistence of the activation after Sephadex chromatography render unlikely the possibility that activators or inhibitors play a significant role in the stimulation of phosphofructokinase by serum. In addition to serum, epidermal growth factor and insulin also enhance the activity of phosphofructokinase. The activation occurs in the presence of 30 microgram/ml of cycloheximide. It is not the result of a high glycolytic flux because drugs (dinitrophenol and oligomycin) that interfere with ATP synthesis and are potent stimulators of glycolysis in intact cells fail to increase phosphofructokinase activity and because the growth-promoting factors increase the enzyme activity in glucose-free medium. Thus, the activation of phosphofructokinase activity appears specifically related to the action of growth-promoting factors and it may offer an experimental system to investigate the chemical signals or cellular conditions, or both, that lead to rapid cell proliferation.