The regulatory kinetic properties of phosphofructokinase partially purified from the livers of C57BL/KsJ mice were studied. The fructose 6-phosphate saturation curves were highly pH dependent. At a fixed MgATP concentration (1 mM), allosteric kinetics was observed in the range of pH studied (7.3 to 8.3) and the S0.5 values for fructose 6-phosphate decreased by about 0.2 to 0.3 mM for each 0.1-unit increment in pH. Allosteric effects on the sigmoidal response to fructose 6-phosphate: activation by AMP, NH4+, and glucose 1,6-bisphosphate, inhibition by MgATP2-, and synergistic inhibition between ATP and citrate, were all present at pH 8.0 to 8.2. Comparative kinetic studies with liver phosphofructokinase isolated from both the normal (C57BL/KsJ) and the genetically diabetic (C57BL/KsJ-db) mice of 9 to 10 and 15 to 16 weeks of age showed that the enzyme from the livers of diabetic mice exhibited decreased activity at subsaturating concentrations of fructose 6-phosphate. However, phosphofructokinase isolated from the livers of normal and genetically diabetic mice of 4 to 5 weeks of age showed no difference in kinetic properties. Thus, there appears to be a correlation between the change in properties of liver phosphofructokinase and the expression of hyperglycemia and obesity in the genetically diabetic mice. The decreased activity of liver phosphofructokinase in the older diabetic animals may well be one of the causes of the increased blood glucose levels. The results are also discussed in a general context with regard to the possible role of phosphofructokinase in the regulation of hepatic gluconeogenesis.