The binding of various regulatory ligands and substrates to the fructose bisphosphate activated pyruvate kinase from Escherichia coli has been studied at equilibrium. The allosteric activator, fructose bisphosphate, and the substrate phosphoenolypyruvate bind in a cooperative manner to the enzyme. There is one site for each of these ligands per monomer. In the presence of fructose bisphosphate the binding of phosphoenolpyruvate follows an absorption isotherm, i.e., all homotropic interactions of the substrate are lost. In reciprocal experiments, however, both phosphoenolpyruvate and KCl are required in order to facilitate binding of the activator. The allosteric inhibitors of pyruvate kinase, ATP, succinyl-CoA, and GTP compete on the enzyme surface with the binding of the activator, fructose bisphosphate, Inhibitor pairs such as ATP and succinyl-CoA together bring about not cooperative but only additive inhibition of the binding of the activator. The nucleotide substrate GDP and the allosteric inhibitor GTP have in contrast to the activator two seemingly noninteracting sites on each monomer. In the saturating presence of fructose bisphosphate, however, binding of GDP and possibly also of GTP occurs at only one site on each monomer. Magnesium ions inhibit binding of GDP and GTP. KCl which is an activator of the enzyme along with its analogues, such as ammonia, thallium, rubidium, etc., enhances the binding of phosphoenolpyruvate but not of the nucleotides or fructose bisphosphate. The data are analyzed on the basis of a two-site model, where the substrate and fructose bisphosphate bind to one conformation and the inhibitors to the other.