Metallothioneins (MTs) constitute a universal family of polymorphic, ubiquitous small Cys-rich metal-binding polypeptides that in mammals are represented by four highly similar isoforms (MT1 to MT4). MT1 and MT2 have generally been considered as equivalent proteins, so that they are commonly referred to as MT1/MT2. However, transcription data have suggested a differential behavior for both gene products. In the present study, the metal binding abilities of mouse MT2 (mMT2) with divalent (Zn(ii) and Cd(ii)) and monovalent (Cu(i)) ions were analyzed and compared to those of the mouse MT1 (mMT1) isoform, previously determined using the same methodological approach. The comprehensive consideration of all the results obtained in this work experimentally demonstrates that the mMT2 isoform exhibits metal ion binding abilities distinct from those of mMT1, with a clear preference for Zn(ii) coordination, if compared to Cu(i) or even to Cd(ii). This is in full agreement with the gene expression regulation pattern for the MT1 and MT2 genes, as well as with the hypothesized preferential role of mMT2 in Zn(ii) homeostasis mechanisms, while MT1, possibly differentiated from a most recent duplication event in the mammalian MT gene cluster, would have evolved to detoxify Cd(ii), and probably other divalent metal ions. © 2013 The Royal Society of Chemistry.