BACKGROUND: Doxorubicin causes a chronic cardiomyopathy of unknown pathogenesis. We investigated whether acquired defects in mitochondrial DNA (mtDNA) and interconnected respiratory chain dysfunction may represent a molecular mechanism for its late onset. METHODS AND RESULTS: Rats were treated weekly with intravenous doxorubicin (1 mg/kg) for 7 weeks, starting at 11 weeks of age (group B). Controls received saline. Group C received doxorubicin identically to group B, but the course was started at 41 weeks of age. All rats were killed at week 48. Doxorubicin was also injected once, either 6 days (group D) or 2 hours (group E) before euthanasia. Heart and skeletal muscle were examined. Only group B rats developed a significant clinical, macroscopic, histological, and ultrastructural cardiomyopathy. Group B hearts had the lowest cytochrome c oxidase (COX) activity (24% of controls; P=0.003), the highest citrate synthase activity (135% of controls; P=0.005), and the highest production of superoxide. In group B, the respiratory subunit COXI, which is encoded by mtDNA, was reduced (P<0.001), as was mtDNA (49% of controls, P<0.001). Group C hearts differed from group B in their lower cardiomyopathy score (P=0.006), higher COX activity (P=0.02), and higher mtDNA content (P=0.04). Group B and to a lesser extent group C hearts contained deleted mtDNA. There was no detectable mitochondrial toxicity in group D and E hearts or in skeletal muscle. CONCLUSIONS: In doxorubicin cardiomyopathy, mtDNA alterations, superoxide, and respiratory chain dysfunction accumulate long-term in the absence of the drug and are associated with a late onset.