Abstract The phosphofructokinase reaction was studied at pH 8 with a free magnesium ion concentration of 4 mm. Under these conditions, inhibition by ATP and cooperativity in fructose 6-phosphate kinetics are not apparent. Plots of reciprocal velocity versus the reciprocal of the concentration of either substrate at different fixed concentrations of the other substrate yield intersecting lines for both the forward and reverse reactions. The Michaelis constants for fructose 6-phosphate and MgATP are 21 and 20 µm, respectively, and the turnover number is 8.6 x 104 moles min-1 per 3.8 x 105 g of enzyme, while the Michaelis constants for MgADP and fructose diphosphate are 20 µm and 0.42 mm, respectively, and the turnover number for the reverse reaction is 3.7 x 103 moles min-1 per 3.8 x 105 g of enzyme. MgADP is a competitive inhibitor versus MgATP, with an inhibition constant, 0.11 mm, independent of fructose 6-phosphate concentration. The other product inhibition patterns, MgADP versus fructose 6-phosphate, and fructose diphosphate versus either MgATP or fructose 6-phosphate, are all noncompetitive. No isotopic exchange of fructose 6-phosphate with fructose diphosphate could be detected in the absence of nucleotides. In the absence of sugar phosphates, the isotopic exchange of ATP with ADP proceeded at less than 1% of the rate of the over-all reaction. d-Arabinose 5-phosphate was found to be a competitive inhibitor of both fructose 6-phosphate and fructose diphosphate and noncompetitive relative to both MgATP and MgADP. MgGDP, an alternative substrate in the reverse direction, was competitive with MgADP with respect to ATP production and noncompetitive relative to fructose diphosphate. These results are most consistent with a sequential mechanism in which the order of addition of substrates is random and is approximated kinetically by the rapid equilibrium assumption. The equilibrium constant for the phosphorylation of free fructose 6-phosphate by MgATP to produce free fructose diphosphate and MgADP is 1.2 x 103 at pH 8 and 30°. Thus the free energy of hydrolysis of the 1-phosphate in fructose diphosphate is identical with that of glucose 6-phosphate.