Metallothioneins (MT) are small cysteine-rich proteins, expressed in many life forms. They are involved primarily in the metabolism of zinc and copper, and in metal detoxification processes. Metallothionein-3 is a mammalian brain-specific MT, which is down-regulated in Alzheimer's disease brains. In this report, we describe a new procedure for purification of recombinant human apo-MT-3 by three steps, size exclusion at neutral pH, followed by cation-exchange and reverse-phase HPLC, both at low pH. Purified apo-MT-3 was reconstituted with seven Zn(2+) ions and reconstitution products were analyzed with electrospray ionization mass spectrometry. The mass spectrum of reconstituted ZnMT-3 was identical with that of native ZnMT-3 isolated by size exclusion chromatography proving the efficiency of the reconstitution process. It showed that ZnMT-3 exists in solution as a dynamic mixture of several metalloforms, where the main metalloform is Zn(7)MT-3 and minor forms are Zn(6)MT-3 and Zn(8)MT-3.