Metallothioneins (MT) are low molecular weight, cysteine-rich, metal-binding proteins that are thought to play key roles in detoxification and Zn homeostasis. Four metallothionein isoform families, termed MT 1-4, have been identified in mammalian species. The majority of studies on MT have focused on the biochemical properties of the widely expressed MT-1 and MT-2 and, in comparison, few studies have investigated the metal binding characteristics of the neuronal-specific MT-3. While the function of MT-3 in neurons is not fully understood, a better understanding of the biochemical properties of MT-3 and its relationship with the other MT isoforms may aid in determining the role of this isoform in Zn homeostasis in the brain. In the current study, different MT isoforms, each labeled with a unique stable isotope of Zn, have been used to study the interaction of Zn between the MT isoforms and to study their ability to donate metal in transfer reactions.