Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules, thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three- primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations in human disease and ageing.