The structure of four new MT (metallothionein) genes of Tetrahymena thermophila and T. pigmentosa were characterized. The MT-2 genes from the two species are very similar, differing by 10 out of 2259 sequenced nucleotides, and the deduced amino acid sequences are identical. The MT-1 genes from T. pigmentosa and T thermophila are also very similar, differing only by 3 nucleotides in the 5'-UT region. The promoter regions contain a TATA box and many stretches partially matching some regulatory elements such as metal-responsive (MREs), antioxidant-responsive (AREs), a CAAT box, a G-box, and AP1 and ACE-1 binding sites. The related coding and amino acid sequences were compared with those previously sequenced in Tetrahymena. This analysis revealed two independent events of duplication occurring in Cd- (MT-1 and MTT1) and Cu- (MT-2) induced MTs. This evolutionary pathway also explains the unusual length of these proteins, which are much longer than many MTs studied so far. Additionally, the orthology and paralogy relationships of the various MTs are presented. Finally, on the basis of phylogenetic analyses of Tetrahymena MTs, two evolutionary hypotheses are proposed.