Replication-competent HIV, as well as HIV-1 DNA, has been detected in CD4 T cells and in monocytes during antiretroviral therapy (ART), indicating that these cells could represent an important viral reservoir. We measured HIV-1 DNA in monocytes and CD4 T cells in patients undergoing transient therapy interruption (TTI), to establish the dynamic of HIV-1 DNA burden and to find possible correlations with immune restoration and re-establishment of virological control after ART resumption. In most patients CD4 depletion and viral load rebound followed TTI. Rapid resumption of virological and immunological control was achieved after ART reintroduction. After TTI, in most cases a transient increase of both monocyte and CD4 HIV-1 DNA burden was observed. After ART reintroduction, both CD4 T cell and monocyte HIV-1 DNA copy number decreased, reaching baseline levels at the end of observation. At this time monocyte HIV-1 DNA burden was always undetectable, while CD4 T cell HIV-1 DNA burden was lower than at baseline. As CD4 T cell HIV-1 DNA values are independently associated with CD4 depletion, the increase of HIV-1 DNA burden in these cells after TTI is presumably due to acute infection, causing cell death. This is also supported by the pattern of 2-LTR appearance in these cells after TTI. HIV-1 DNA burden in monocytes and CD4 T cells show high correlation, suggesting reciprocal re-feeding of two cell populations. Repopulation by HIV these cells after TTI is temporary, and no significant changes of HIV-1 DNA burden were observed after ART resumption respect to pre-TTI period.