Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation 1, 2, 3, 4 and may also have a role in ageing and acquired diseases of old age 5. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA (Refs 6, 7). Using a loxP-flanked Tfam allele (Tfam loxP; ref. 8) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter 9, 10, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome 1, 2, 3, 4. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.