We report the cloning and characterization of two new metallothionein (MT) genes (TrosMTT1 and TrosMTT2), isolated as cDNAs, from the ciliated protozoa Tetrahymena rostrata. The TrosMTT1 inferred protein has been identified as a CdMT and included into the 7a subfamily of Tetrahymena MTs, while TrosMTT2 has been identified as a CuMT (including it into 7b subfamily), due to its similarity to TpigMT-2 and its significant induction by copper. TrosMTT1 protein sequence reveals a remarkably regular and hierarchical modular organization, as it is known for other Tetrahymena CdMTs, showing a bi-modular structure. TrosMTT2 presents a structural organization based on CKCX(2-5)CKC repeats, like it occurs in other Tetrahymena CuMTs, indicating that an evolutionary history based on intra-gene duplications might be also possible. Both are also multi-stress-inducible genes because they are induced by other heavy metals and stressors, as it has been shown by quantitative real-time RT-PCR. It is the first time that the gene expression of a putative Tetrahymena CuMT is analyzed by quantitative PCR, confirming it as a CuMT. These two new Tetrahymena MTs complete, at present, the actual view of this protein superfamily, and corroborate the unique features of ciliate MTs. Furthermore, both, a comparative analysis of relative gene expression values obtained by quantitative RT-PCR on other Tetrahymena MT genes and an analysis of the different Tetrahymena MTs based on the different Cys clusters of these proteins are carried out, which show an update view of Tetrahymena MT gene family.