Defects in myocardial bioenergetics have been reported in patients with cardiomyopathy but their molecular basis and role in pathophysiology remain unclear. We sought to establish a molecular basis for cardiac mitochondrial respiratory enzyme abnormalities frequently present (75%) in a group of 16 children (including 2 neonates) with end-stage cardiomyopathy. Decreased specific activity levels were found in complexes I, III, IV and V but not in II, the only complex that is entirely nuclear encoded. Sequence analysis of cardiac mtDNA revealed 4 patients harbouring heteroplasmic mtDNA mutations in cytb, tRNA(Arg), and ND5 at highly conserved positions. These mutations were present neither in controls nor in patients without enzymatic defect. In addition, 4 patients exhibited marked reduction in cardiac mtDNA levels. The basis for respiratory enzyme abnormalities can be explained in a subset of our patients as a result of either pathogenic mtDNA mutation or depletion. Patients harbouring both DNA and enzymatic defects fulfil rigorous criteria defining mitochondrial cardiomyopathy.