The regulatory properties of citrate on the activity of phosphofructokinase (PFK) purified from rat-kidney cortex has been studied. Citrate produces increases in the K0.5 for Fru-6-P and in the Hill coefficient as well as a decrease in the Vmax of the reaction without affecting the kinetic parameters for ATP as substrate. ATP potentiates synergistically the effects of citrate as an inhibitor of the enzyme. Fru-2,6-P2 and AMP at concentrations equal to Ka were not able to completely prevent citrate inhibition of the enzyme. Physiological concentrations of ATP and citrate produce a strong inhibition of renal PFK suggesting that may participate in the control of glycolysis in vivo.