Anthracyclines are antitumor agents whose main clinical limitation is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage, and whether this damage is linked to a signaling pathway culminating in cell death. H9C2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed utilizing the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment, in contrast to the complete repair observed in H₂O₂-treated myocytes following removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential following p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxy guanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.