Metallothionein (MT) has been shown to be an effective protector against DOX-induced cardiomyopathy, however the involved precise mechanisms are still unknown. The present study was undertaken to clarify whether the inhibition of superoxide generation and related nitrosative damage were involved in the metallothionein attenuation of DOX-induced cardiac injury. MT-I/II null (MT-/-) mice and corresponding wild-type mice (MT+/+) were pretreated with either saline or zinc (300 micromol/kg, s.c., once a day for 2 days) prior to a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and samples were collected for further analyses. DOX caused remarkable cardiac damage in both MT+/+ and MT-/- mice as demonstrated by biochemical and histopathological alterations. Zinc pretreatment significantly increased the cardiac MT levels and therefore inhibited the cardiac toxic effects of DOX only in MT+/+ mice, but not in MT-/- mice. Furthermore, elevated formation of superoxide and peroxynitrite were obviously observed after DOX treatment, while these elevation were prevented by MT induction by zinc in MT+/+ mice, but not in MT-/- mice. These findings suggest that metallothionein induction by zinc exhibits protective effects on the cardiac toxicology of DOX, which might be mediated through the prevention of superoxide generation and related nitrosative impairment.