The specific activity and/or the allosteric behavior of rabbit muscle phosphofructokinase is dependent on several factors including (1) the method of assay, and (2) the concentration, pH, and temperature of the enzyme solution from which dilution is made into the assay mixture. These observations suggest that quantitative interpretation of the allosteric characteristics of this enzyme may be in error because of lack of control of any or all of these factors. In some buffer systems, such as imidazole at pH 7, instability of the enzyme in the assay leads to further complications in the interpretation of previous studies. The results of the present paper show that under specific conditions it is possible to obtain allosteric kinetic data from which quantitative interpretations can be made. This is best accomplished by performing experiments in phosphate buffer and coupling the reaction through pyruvate kinase and lactic dehydrogenase. The experiments must be carried out either in the presence of excess fructose 1,6-bisphosphate or fructose diphosphatase in order to control the level of the activator fructose 1,6-bisphosphate. Under such conditions, the allosteric kinetic behavior observed at pH 6.5 does not appear to be a consequence of polymerization between an active (four subunit) and inactive (two subunit) form of the enzyme, but is inherent in the active form.