This paper reports on the characterization of the metal binding abilities of mammalian MT4 and their comparison with those of the well known MT1. Heterologous Escherichia coli expression in cultures supplemented with zinc, cadmium, or copper was achieved for MT4 and for its separate alphaMT4 and betaMT4 domains as well as for MT1 and its alphaMT1 domain in cadmium-enriched medium. The in vivo conformed metal complexes and the in vitro substituted zinc/cadmium and zinc/copper MT4 aggregates were characterized. Biosynthesis of MT4 and betaMT4 in Cd(II)-supplemented medium revealed that these peptides failed to form the same homometallic species as MT1, thus appearing less effective for cadmium coordination. Conversely, the entire MT4 and both of its domains showed better Cu(I) binding properties than MT1, affording Cu(10)-MT4, Cu(5)-alphaMT4 and Cu(7)-betaMT4, stoichiometries that make the domain dependence toward Cu(I) clear. Overall results allow consideration of MT4 as a novel copper-thionein, made up of two copper-thionein domains, the first of this class reported in mammals, and by extension in vertebrates. Furthermore, the in silico protein sequence analyses corroborated the copper-thionein nature of the MT4 peptides. As a consequence, there is the suggestion of a possible physiological role played by MT4 related with copper requirements in epithelial differentiating tissues, where MT4 is expressed.