The two-domain (beta alpha) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain beta alpha-rhMT and the isolated beta-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd(3)-beta-rhMT and Cd(7)-beta alpha-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd(4)-beta- and Cd(8)-beta alpha-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins.