The clinically approved antioxidant cardioprotective agent dexrazoxane (ICRF-187) was examined for its ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle, both through reductive activation to its semiquinone form, and by the production of hydroxyl radicals mediated by its complex with iron. Hydrolyzed dexrazoxane metabolites prevent site-specific iron-based oxygen radical damage by displacing iron from doxorubicin and chelating free and loosely bound iron. The mitochondrial stain MitoTracker Green FM and doxorubicin were shown by epifluorescence microscopy to accumulate in the myocyte mitochondria. An epifluorescence microscopic image analysis method to measure mitochondrial damage was developed using the mitochondrial membrane potential sensing ratiometric dye JC-1. This method was used to show that dexrazoxane protected against doxorubicin-induced depolarization of the myocyte mitochondrial membrane. Dexrazoxane also attenuated doxorubicin-induced oxidation of intracellular dichlorofluorescin. Annexin V-FITC/propidium iodide staining of myocytes was used to demonstrate that, depending on the concentration, doxorubicin caused both apoptotic and necrotic damage. These results suggest that doxorubicin may be cardiotoxic by damaging the mitochondria and dexrazoxane may be protective by preventing iron-based oxidative damage.