Mitochondrial DNA (mtDNA) deletions increase in abundance with age in many tissues, however, their calculated low levels (usually < 0.1%) in samples from tissue homogenates containing thousands of cells argue against physiologic significance. Through the analysis of defined numbers of cells (skeletal muscle fibers) from rhesus monkeys, we report that the calculated abundance of specific mtDNA deletions is dependent upon the number of fibers analyzed: as the number of fibers decreases, the calculated deletion abundance increases. Also, most mtDNA deletions appear to occur in a mosaic pattern, varying from cell to cell in size, number and abundance. These data support the hypothesis that mtDNA deletions can focally accumulate to high levels contributing to declines in mass and function of aging skeletal muscle.