Nucleoside analogue use is often related to mitochondrial DNA (mtDNA) depletion, but mitochondrial function is preserved in most asymptomatic patients. We determined whether homeostatic mechanisms are able to compensate for this mtDNA depletion in patients receiving stavudine plus didanosine (d4T + ddI), an antiretroviral combination with great in vitro and in vivo capacity to decrease mtDNA. We included 28 asymptomatic HIV-infected individuals: 17 subjects (cases) on a first-line antiretroviral regimen consisting of d4T + ddI as the nucleoside backbone plus nevirapine or nelfinavir for at least 6 months (mean: 16 ± 8 months) and 11 naive subjects (controls). We assessed the following in peripheral blood mononuclear cells: mitochondrial mass by citrate synthase activity, mtDNA content by real-time polymerase chain reaction, cytochrome c oxidase subunit II (COX-II) expression by Western blot analysis, and COX activity by spectrophotometry. The mitochondrial mass and mtDNA content of cases decreased when compared with controls, whether normalized per cell or per mitochondrion. Conversely, COX-II expression and COX activity were similar in cases and controls. COX-II expression was constant and independent of the mtDNA content, whereas it was closely related to COX activity. We concluded that treatment with dd4T + ddI is associated with decreased mitochondrial mass and mtDNA content but that COX-II expression and COX activity remain unaltered. These data suggest that upregulatory transcriptional or posttranscriptional mechanisms compensate for mtDNA depletion caused by d4T + ddI before profound mtDNA depletion occurs.