Post-mitotic tissues such as skeletal muscle develop a tissue bioenergy mosaic during the process of normal aging that eventually culminates into a bioenergetically diverse tissue containing cells ranging in their oxidative phosphorylation capacity from normal to grossly defective. The mosaic is postulated to develop continuously from birth with the relative proportions of cytochrome c oxidase (COX) proficient (positive) and COX deficient (negative) muscle fibers differing dramatically as a function of age. Generally, young individuals only display the rare fiber deficient in COX activity while aged individuals show a significantly higher proportion of negative fibers. There appears to be a random element governing which cells will be affected. Consequently, adjacent cells within a given tissue may exhibit vastly differing COX activities. Multiple mitochondrial DNA (mtDNA) deletions also appear to accumulate in skeletal muscle, similarly displaying a dramatic disparity as a function of age. Our previous findings have indicated that the accumulation of multiple mtDNA deletions, along with a concurrent decrease in wild-type mtDNA, strongly correlates with the age-associated decrease in COX activity observed in skeletal muscle. Although no definitive associations were established at the cellular level, an important prediction arose from this study. Cells that accumulate large numbers of mitochondrial mutations and have reduced levels of full-length mtDNA would be expected to be severely affected and show reduced COX activity as a consequence. Cells that accumulate fewer mutations or retain adequate amounts of wild-type mtDNA would be predicted to be less affected or even retain normal oxidative metabolism. In order to establish a link associating COX activity to the status of mtDNA within individual fibers, we developed single cell extra-long PCR (XL-PCR). The procedure was used to assess the relative concentration of full-length mtDNA with respect to any mtDNA deletions detected in individual human skeletal muscle fibers of 'pre-established' COX activity. Single cell XL-PCR analysis of COX positive fibers dissected from a 5-year old and 90-year old individual showed that 80% or more of the fibers contained full length mtDNA and few, if any, mtDNA rearrangements. COX deficient or COX intermediate fibers taken from the same individuals, by contrast, depicted a heterogeneous population of rearranged mtDNA species with no detectable full-length mtDNA. The data presented here indicates that COX deficient muscle fibers extracted from individuals, regardless of age, were accompanied by extensive mtDNA rearrangements and reduced levels of full-length mtDNA. This provides compelling evidence linking mtDNA mutations to COX activity decline in skeletal muscle and has important implications when considering the molecular basis of the aging process.