Background: HIV infection per se and HAART can alter mitochondrial functionality, leading to a decrease in mitochondrial DNA content. Objective: To evaluate whether treatment interruption monitored by CD4 cell count can restore mitochondrial DNA content in peripheral blood lymphocytes. Methods: Mitochondrial DNA content was measured in platelet-free CD4 and CD8 T cells by real-time polymerase chain reaction; flow cytometry was used to identify and quantify activated CD4 and CD8 T lymphocytes. Results: The 30 patients had been treated for a mean of 107 months (range, 27-197). Median CD4 cell count at discontinuation was 702 cells/mu l (range, 547-798). Median observational time from HAART discontinuation was 11.3 months (range, 4-26). Discontinuation of treatment provoked significant increases in mitochondrial DNA in CD8 T cells, which started only 6 months after therapy cliscontinuation [5.12 copies/ cell per month from 0 to 6 months (P = 0.3 7) and 2 6.96 copies/cel I per month from 6 to 12 months (P < 0.0001)]. Conclusions: This study is the first showing that mitochondrial DNA content can increase in peripheral blood lymphocytes during treatment interruption, but only after at least 6 months of interruption. Consequently, interruptions of shorter periods, whether by clinician or patient decision, are unlikely to allow restoration of mitochondrial DNA and so decrease HAART-related toxicity.