The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd - and the Cu - thioneins. Ciliates harbor the largest MT gene/protein family reported so far, i n- cluding 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. I n Te t- rahymena thermophila , three MTs (MTT1, MTT3 and MTT5) were considered Cd - thioneins and two (MTT2 and MTT4) Cu - thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal - binding abilities of the five MTT pr oteins were characterized, to obtain information about the folding and stability of their cognate - and non - cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn 2+ - , Cd 2+ - or Cu + - complexes, which were analyzed by electrospray mass spectrometry (ESI - MS), circular dichroism (CD), and UV - vis spectrophotometry. Among the Cd - thioneins, MTT1 and MTT5 were optimal for Cd 2+ coordination, yielding unique Cd 17 - and Cd 8 - com plexes, respectively. When binding Zn 2+ , they rendered a mixture of Zn - species. Only MTT5 was capable to coordinate Cu + , although yielding heteronuclear Zn - , Cu - species or highly unstable Cu - homometallic species. MTT3 exhibited poor binding abilities both for Cd 2+ and for Cu + , and although not optimally, it yielded the best result when coordinating Zn 2+ . The two Cu - thioneins, MTT2 and MTT4 isoforms formed homometallic Cu - complexes (major Cu 20 - MTT) upon synthesis in Cu - supplemented hosts. Contrarily, they we re unable to fold into stable Cd - complexes, while Zn - MTT species were only recovered for MTT4 (major Zn 10 - MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd - and Cu - thioneins, a nd globally, they can be classified from Zn/Cd - to Cu - thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been i n- ternal tandem duplica tions, presence of doublet and triplet Cys patterns in Zn/Cd - thioneins, and o p- timization of site specific amino acid determinants (Lys for Zn/Cd - and Asn for Cu - coordination).