We report here that Escherichia coli pyrophosphatase aggregates in the presence of millimolar Cd2+. This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd2+ concentration. Aggregation was enhanced by Mg2+, the natural cofactor of pyrophosphatase, and Mn2+. Mutations at the intersubunit metal-binding site had no effect, whereas mutation at Glu139, which is part of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme molecules, suppressed aggregation. These findings indicate that aggregation is affected by Cd2+ binding to the peripheral metal-binding site, probably by strengthening intermolecular Trp149–Trp149′ stacking interactions.