In order to systematically characterize age-related changes in the mtDNA content of various tissues during aging, we analyzed the mtDNA content of eight tissues from mice at five different ages from young to senescent by quantitative real-time PCR analysis. Obvious variations of mtDNA content among the tissues were detected: There was a 20-fold range in 2-week-old mice and a 50-fold range in 15-month-old mice. The mtDNA contents of the heart, lung, kidney, spleen and skeletal muscle increased gradually with age, whereas those of bone marrow and brain showed no age-related pattern. The expression patterns of mitochondrial transcription factor A (mtTFA) and mitochondrial single-strand DNA binding protein (mtSSB), possible regulatory factors of the mtDNA copy number, were not necessarily linked with the age-related pattern of the mtDNA content, suggesting the existence of other factors that affect the mtDNA content. The Western blot analysis of mtDNA-encoded cytochrome c oxidase subunit III (MTCO3) demonstrated that the expression levels of this protein in the heart and skeletal muscle increase with age in parallel with the mtDNA content. These findings confirm that the mtDNA content of tissues changes during aging.