The kinetic behaviour of different preparations of yeast phosphofructokinase is described, predominantly regarding the interactions between substrate-and effector-binding sites.The substrate-binding site for ATP reacts with I T P and UTP too, whereas the ATP-inhibitor site possesses higher specsty: other nucleoside triphosphates do not compete with ATP for this site.Binding of protons to the enzyme produces a decrease in cooperativity towards fructose-6-phosphate with ATP as well as with ITP and UTP as phosphate donors, accompanied by a lowering of the fructose-6-phosphate concentration necessary for half-maximal activity. At the same time the inhibitory action of ATP is descreased.AMP is bound t o an effector site evidently different from the ATP-inhibitor site.With ITP as phosphate donor, AMP shows no activating effect. AMP decreases the apparent K , value for fructose-6-phosphate too without affecting cooperative interactions. Magnesium and ammonium ions are necessary for full enzymatic activity. The latter increases maximal activity and decreases the half-maximal concentration of fructose-6-phosphate. Free ATP seems to be a stronger inhibitor for yeast phosphofructokinase than the magnesium-ATP-chelate.