The rainbow trout hepatoma (RTH) cell line responds to heavy metals such as zinc and cadmium by synthesizing the ubiquitous thiol-rich protein metallothionein (MT). From this cell line we have isolated two full-length cDNA clones, tMT-A and tMT-B, which encode two similar but distinct trout MTs. The clones were isolated by cross-homologies between the trout MT mRNAs and a human MT riboprobe. Clones tMT-A and tMT-B code for proteins of 61 and 60 amino acids, respectively; the one extra amino acid in tMT-A is due to an apparent insertion at position 31 of the protein. There are also two other amino acid changes between the two isoforms. Overall, the coding regions show extensive homologies to mammalian MTs, especially at the cysteine residues and at a core sequence at the boundary of the two domains. However, closer examination reveals a number of significant differences in positions usually invariant in the mammalian MTs. Northern blot analysis of RNA from metal-treated RTH cells demonstrated MT-mRNA is induced to high levels by zinc, low levels by cadmium, and minimally by copper. In contrast, intraperitoneal injections of rainbow trout demonstrated that all three metals induce MT-mRNA to comparable levels in the liver. Southern blot analysis of trout DNA cleaved with three restriction enzymes suggests that the trout family of MT genes is probably limited to these two members.