Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn and Cu binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn binding was measured by chelation titrations of ZnMT-3, while Cu binding was measured by Zn displacement from ZnMT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant () and the change in enthalpy (Δ) and entropy (Δ) for these metal ions binding to MT-3. Zn was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of ZnMT-2 with Cu revealed that both MT isoforms have similar Cu affinities and binding thermodynamics, indicating that Δ and Δ are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu -thiolate clusters when Cu displaces Zn under physiological conditions. Comparison of the Zn and Cu binding thermodynamics reveal that enthalpically-favoured Cu, which forms Cu -thiolate clusters, displaces the entropically-favoured Zn. These results provide a detailed thermodynamic analysis of d metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.