Tenofovir disoproxil fumarate (TDF) has been reported to be free of adverse effects on mitochondria. We evaluate the effects of the introduction of TDF in a didanosine (ddI)-based highly active antiretroviral therapy (HAART) on mitochondrial DNA (mtDNA) content, mitochondrial mass (MM), and cytochrome c oxidase (COX) activity of the oxidative phosphorylation (OXPHOS) system over a 12-month period. Forty-four asymptomatic HIV patients with undetectable viral load receiving a ddI-based HAART were recruited and switched to ddI plus TDF (ddI + TDF) and nevirapine (n = 22) or maintained with the same baseline ddI-based HAART scheme (n = 22). Peripheral blood mononuclear cells were obtained at 0, 6, and 12 months. COX activity and MM were determined by spectrophotometry and the mtDNA content by quantitative real-time PCR. The mtDNA content showed a progressive decrease over the 12-month period of the study for the two groups with respect to baseline, with such a decrease statistically significant only in the ddI + TDF group (55% decrease, p < 0.001). In addition, the decrease of mtDNA content over time was statistically different between both groups (p < 0.001). Consistently, MM and COX activity decreased significantly at 12 months with respect to baseline only in the ddI + TDF group (28% decrease for MM, p < 0.05; 47% decrease for COX activity, p < 0.001). We conclude that switching to a HAART regimen containing ddI + TDF is associated with evolutive mitochondrial damage expressed as mtDNA depletion, loss of MM, and decrease in COX efficiency. The particular relevance of either ddI, TDF, or any interaction between them in such a mitochondrial dysfunction remains to be established. © Mary Ann Liebert, Inc.