Previous studies have shown that cardiac-specific metallothionein (MT)-overexpressing transgenic mice are highly resistant to acute cardiotoxicity induced by doxorubicin (DOX), a most effective anticancer agent. However, cumulative dose-dependent chronic cardiotoxicity attributable to long-term administration of DOX is a significant clinical problem. Because MT is a potent antioxidant and oxidative stress is critically involved in DOX-induced heart injury, the present study was undertaken to test the hypothesis that MT also provides protection against DOX chronic cardiotoxicity. Transgenic mice containing high levels of cardiac MT and nontransgenic controls were treated with a cumulative dose of 40 mg/kg of DOX in 10 equal i.v. injections over a period of 7 weeks. Three weeks after the last injection, the mice were killed for an analysis of cardiotoxicity. As compared with nontransgenic controls, DOX-induced cardiac hypertrophy was significantly inhibited in the transgenic mice. Light microscopic examination revealed that DOX-induced myocardial morphological changes were markedly suppressed or almost eliminated in the transgenic mice. Under electron microscopy, extensive sarcoplasmic vacuolization and severe disruption of mitochondrial fine structure were observed in nontransgenic cardiomyocytes, but almost no sarcoplasmic vacuolization was observed, and the mitochondrial structural changes were almost completely prevented in the transgenic cardiomyocytes. The results thus indicate that MT elevation is a highly effective approach to prevent chronic cardiomyopathy attributable to DOX. This study also suggests that oxidative stress is critically involved in the DOX-induced chronic cardiotoxicity.