Previous studies using a cardiac-specific metallothionein-overexpressing transgenic mouse model have demonstrated that metallothionein protects the heart from doxorubicin toxicity. The present study was undertaken to determine cellular and subcellular distribution of metallothionein and located the antioxidant action of this protein in the transgenic heart. Using light microscopic immunoperoxidase method, it was identified that the overexpressed metallothionein is localized exclusively in cardiomyocytes. The electron microscopic immunogold method revealed that elevated metallothionein is in nucleus, myofibers, and sarcoplasm. In contrast with these distributions, metallothionein in nontransgenic myocardium was undetectable by immunoperoxidase light microscopy and was seldom found in nucleus and myofibers by immunogold electron microscopy. Treatment with doxorubicin induced cytoplasmic vacuolization and severe damages in myofilaments and nucleus in nontransgenic myocardium. The most prominent injury, however, occurred in mitochondria, including striking size and shape changes, focal swelling and loss of cristae. These damages were rarely found in the doxorubicin-treated transgenic myocardium. In particular, the internal morphology of mitochondria was maintained essentially normal, although metallothionein was not localized in this compartment in transgenic hearts. This study thus demonstrates that although the subcellularly localized action of metallothionein is important, it also plays a significant role in protection against oxidative injury by doxorubicin in remote organelles.