We mapped the distribution and expression of wild-type and deleted mitochondrial DNA (mtDNA) molecules in skeletal muscle fibers of patients with mitochondrial disease. We show that ragged red fiber segments, which are characteristic histological features of these myopathies, represent focal accumulations of mitochondria containing predominantly deleted mtDNAs and that the mutant genomes are absent or extremely rare in normal fiber segments. This suggests that the mtDNA mutations play a direct role in focal mitochondrial accumulation. Although levels of wild-type mtDNAs and mRNAs in ragged red fiber segments are near normal, mitochondrial function, as revealed by cytochrome oxidase cytochemistry, is severely impaired. This suggests that the presence of mutant mtDNAs interferes with the expression of coexisting wild-type mtDNAs in these segments at a posttranscriptional level.