Abstract Because of the requirement for divalent metal ion activation, inorganic pyrophosphatase activity is measured in a complex equilibrium mixture containing free metal ion, free PPi, and metal ion-PPi complexes. Using crystalline yeast inorganic pyrophosphatase the binding to the enzyme of each of these individual components of the equilibrium has been examined. The dissociation constants of the binary enzyme-metal ion complexes are 2, 11, 16, and 800 µm for Mn2+, Zn2+, Mg2+, Ca2+, respectively. In the absence of free metal ions neither PPi nor CaPPi bind to the enzyme; in the presence of excess Ca2+, CaPPi is bound quite strongly (Kd apparent = 7 x 10-9 m). The enzyme has two binding sites for CaPPi per mole of enzyme dimer. The results of these static binding studies are consistent with the kinetic analyses presented in the following papers.