An age-related increase of DNA damage/mutation has been previously reported in human lymphocytes. The high copy number and mutation rate make the mtDNA genome an ideal candidate for assessing damage and to act as a potential biomarker of ageing. In the present study, two assays were developed to evaluate the level of mtDNA(4977) and the accumulation of point mutations with age. A competitive polymerase chain reaction (PCR) methodology incorporating three primers was used to detect and quantify the levels of mtDNA(4977) and a novel heteroduplex reference strand conformational analysis (RSCA) technique was used to analyse the accumulation of point mutations. The assays were applied to an in vitro model of T cell ageing and ex vivo DNA samples from an elderly cohort of subjects and a younger control group. The mtDNA(4977) was detected in all the DNA samples examined but only a very low concentration was observed and no age-related increase or accumulation was observed. No accumulation of point mutations was identified using RSCA within the T cell clones as they were aged or the ex vivo lymphocytes from the elderly cohort. A higher level of variation was observed within the ex vivo DNA samples, verifying the high resolution of RSCA and its ability to identify different mtDNA species, although no correlation with age was observed. The low level of mtDNA damage observed with respect to the ex vivo lymphocyte DNA samples within this study may be due in part to the high turnover of blood cells/mtDNA, which may inhibit the accumulation of genetically abnormal mtDNA that may play a role in immunosenescence. A similar explanation may also apply to the in vitro model of T cell ageing if the vast majority of the cells are replicating rather than entering senescence.