ABSTRACT Lipoatrophy is a prevalent side effect of antiretroviral treatment of human immunodeficiency virus (HIV) infection. Its mechanisms are still disputed but include mitochondrial toxicity and, in particular, mitochondrial DNA (mtDNA) depletion induced by nucleoside reverse transcriptase inhibitors. To obtain an integrated evaluation of the mitochondrial alteration in lipoatrophy, we investigated the DNA, RNA, and protein levels in 15 samples of abdominal subcutaneous adipose tissue from HIV-infected patients with peripheral lipoatrophy and compared the results with those for 15 samples from age- and body mass index-matched controls. The DNA and RNA analyses used PCR-based techniques, while proteins were quantified with enzyme-linked immunosorbent assay and measurement of activities with spectrophotometric assays. Depletion of mtDNA and mtDNA-encoded MT-CO2 mRNA was present, but normal levels of mtDNA-dependent activity (cytochrome c oxidase) and protein (MT-CO2p) showed that it was compensated for. An increase in nuclear-DNA-dependent mitochondrial activities (citrate synthase and malate dehydrogenase) and protein (COX4I1p), as well as transcriptional up-regulation of nuclear-DNA-encoded mitochondrial genes ( COX4I1 and UCP2 ), demonstrated increased mitochondrial biogenesis. However, the expression of the known transcription factors of mitochondrial biogenesis ( TFAM , NRF1 , GABPA , PPARGC1A , PPARGC1B , and PPRC1 ) was normal or decreased. Increased amounts of activated caspase 3 and of DDIT3 mRNA showed the induction of apoptosis and oxidative stress, respectively. The mtDNA content did not correlate with any other mitochondrial parameter. In conclusion, mtDNA content does not appear to be an accurate biomarker of mitochondrial alteration in lipoatrophic adipose tissue. The preservation of mtDNA-dependent mitochondrial functions occurred despite severe mtDNA depletion. The presence of significant oxidative stress and apoptosis did not correlate with the mtDNA content.