The role of metallothionein (MT) in free radical regulation and scavenging was investigated using two fish cell lines, the rainbow trout gonadal (RTG-2) cell line and the chinook salmon embryonic (CHSE-214) cell line. Exposure of RTG-2 cells to H 2 O 2 resulted in upregulation of both MT mRNA and MT protein and was also demonstrated by immunocytochemistry, confirming that MT was regulated by free radicals. We then compared the H 2 O 2 resistance in RTG-2 and CHSE-214 cells following metal treatment with Zn or Cd to induce MT. Comparison of survival of control cells and metal-exposed cells showed that metal treatment, which induced MT, significantly raised the H 2 O 2 tolerance in a dose-dependent manner in RTG-2 cells, while no increased H 2 O 2 resistance was observed in CHSE-214 cells. Transient over-expression of MT in CHSE-214: 59 cells also resulted in a dose-dependent increase in resistance to H 2 O 2 exposure. The raised resistance against H 2 O 2 in metal treated RTG-2 cells as well as transfected CHSE-214: 59 cells strongly demonstrate that MT is involved in the protection against H 2 O 2 and suggest a physiologically important function for MT when cells or whole organisms are exposed to oxidative stress.