Previous findings from our own laboratory have shown that the frequency of occurrence (i.e. the simple presence or absence) of the 3895 bp mitochondrial DNA deletion is increased with increasing sun exposure. The present study has significantly extended this work by developing, validating and then using a quantitative real-time PCR assay to investigate for the first time the actual level (as opposed to the frequency of occurrence) of the 3895 bp deletion in human skin from different sun-exposed body sites and tumours from nonmelanoma skin cancer patients. We investigated the 3895 bp deletion in 104 age-matched split human skin samples taken from various sun-exposed sites defined as usually exposed (n = 60) and occasionally exposed (n = 44) when outdoors. The results clearly show an increased level of the 3895 bp deletion with increasing sun exposure. Specifically, there was a significantly higher level of the deletion in the usually sun-exposed compared to the occasionally sun-exposed skin (P = 0.0009 for dermis, P = 0.008 for epidermis; two-tailed t-test). Our study has also extended previous findings by showing that the level of the 3895 bp deletion is significantly higher in the dermis compared with the epidermis both in the occasionally sun-exposed samples (P = 0.0143) and in the usually sun-exposed skin. (P = 0.0007).