The antioxidant function of metallothionein (MT) was first suggested in the early 1980s. Studies in vitro have revealed that MT reacts directly with reactive oxygen species, including superoxide and hydroxyl radicals and hydrogen peroxide. These reactions have never been demonstrated in intact animal studies. Nevertheless, both pharmacologic and genetic studies have shown that MT functions in protection against oxidative injury in vivo. In particular, the antioxidant function of MT in the heart has been explored extensively. The data gathered from recent studies using a cardiac-specific, MT-overexpressing transgenic mouse model have provided direct evidence to support this physiological role of MT. Under acute and chronic oxidative stress conditions such as treatment with doxorubicin, ischemia-reperfusion, and dietary copper restriction, MT-overexpressing transgenic mouse hearts displayed a marked resistance to the injurious consequences, including biochemical, pathological, and functional alterations. This protective action of MT correlates with its inhibition of reactive oxygen species-induced lipid peroxidation. A critical elucidation of the mechanism of action of MT as an antioxidant in vivo remains to be achieved. However, the combination of recent understanding of the zinc cluster structure of MT and novel molecular genetic approaches has provided the basis for further advancement in this field.