Human metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain, where it plays an important role in the homeostasis of the essential metal ions Cu(+) and Zn(2+). Like other mammalian metallothioneins (MT-1 and -2), the protein contains a M(II)(3)(CysS)(9) and a M(II)(4)(CysS)(11) cluster localized in two independent protein domains linked by a flexible hinge region. However, there is a substantially increased number of acidic residues in MT-3 (11 residues) compared with MT-2 (four residues) which may act as binding ligands for additional metal ions. In this study, the binding of Zn(2+), Ca(2+), and Mg(2+) to human Zn(7)MT-3 and its mutant lacking an acidic hexapeptide insert, Zn(7)MT-3(Delta55-60), was investigated and compared with the binding of Zn(7)MT-2. By using spectroscopic and spectrometric techniques, we demonstrate that one additional Zn(2+) binds with an apparent binding constant (K(app)) of approximately 100 microM to Zn(7)MT-3 and Zn(7)MT-3(Delta55-60), but not to Zn(7)MT-2. The changes in spectroscopic features of metal-thiolate clusters and gel filtration behavior reveal that the formation of Zn(8)MT-3 is immediate and is accompanied by a decrease in the Stokes radius (R(s)). The changes in the R(s) suggest a mutual approach of both protein domains. The fast binding of Zn(2+) is followed by a slow time-dependent protein dimerization. The binding of Zn(2+) to Zn(7)MT-3 is specific as in the presence of Ca(2+) and Mg(2+) only an alteration of the R(s) of Zn(7)MT-3 at substantially higher concentrations was observed. The significance of these findings for the biological role of MT-3 is discussed.