Phosphofructokinase (EC 126.96.36.199) from rabbit liver was purified to homogeneity. Preincubation of enzyme results in nonlinearity of enzyme activity with enzyme concentration. Therefore K0.5 of enzyme for fructose 6 phosphate in the absence or presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both was determined at physiological concentrations of its various effectors by taking the initial rate obtained by adding the enzyme last. They decrease the K0.5 value from 4.1 mM to about 0.2mM. The K0.5 of enzyme for fructose 2,6 bisphosphate was also determined under the above conditions. It is about 4.3ΜM. Transient kinetics of phosphofructokinase at varying concentrations of enzyme in the presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both were studied. It was found that although they decrease t1/2i.e. the time to reach half the maximal steady rate by about 5–8 fold, it was about constant at varying concentrations of the enzyme. These results indicate that fructose 2,6 bisphosphate and polyethylene glycol decrease K0.5 of the enzyme for fructose 6 phosphate not by associating the enzyme to higher aggregates, but by a different mechanism.