Ferritins are iron-storage proteins capable of holding up to 4500 Fe3+ ions within a single water-soluble protein shell made from 24 polypeptide chains. The Glu128Arg/Glu135Arg mutants of Escherichia coli and Rhodobacter capsulatus bacterioferritins are unable to associate into 24-meric structures, with dinners of polypeptide chains being their stable forms. The aerobic addition to these of up to 8-10 or 14-20 Fe2+ ions per dimer, respectively, results in the oxidation of the added Fe2+ to Fe3+. Gel permeation chromatography and sedimentation equilibrium studies confirm that the Fe3+ ions are associated with the polypeptide dinner, and the lack of intense EPR signals from magnetically isolated Fe3+ ions confirms the formation of one or more antiferromagnetically coupled clusters of Fe3+ ions. The effect of Fe3+ chelators on iron-loaded subunit dinners is to remove the Fe3+ from the protein, but to do so slowly, consistent with it not being merely adventitiously associated with protein. These data provide experimental support for the presence of nucleation centers for the mineral cores in bacterioferritins and indicate that these proteins are not simply acting as vessels in which hydrolysis of Fe3+ occurs independent from the protein surface. From analyses of X-ray structures and amino acid sequence comparisons, possible nucleation sites are identified.