During highly active antiretroviral therapy (HAART), HIV-1 can still persist in circulating, resting CD4+ T lymphocytes, lymph node mononuclear cells, and seminal cells of patients despite sustained suppression of plasma viremia to undetectable levels. Sanctuary sites where antiretroviral drug penetration is not optimal may allow local HIV-1 infection of cells within and passing through these tissues. Factors such as imperfect drug adherence due to complicated drug regimens may also result in tissue compartments with suboptimal drug concentrations allowing viral replication. We have examined blood monocytes from HIV-1-infected subjects being effectively treated with HAART to determine virus carriage in these cells. Monocytes were purified from peripheral blood of patients with plasma HIV-1 RNA below 50 copies/mL and who had maintained levels of plasma RNA below detection for 3 months or more. Replication-competent virus could be recovered from the majority of monocyte populations by co-culture with CD8-depleted, PHA-activated, peripheral blood mononuclear cells. Sequencing of the reverse transcriptase and protease genes of the recovered viruses did not reveal resistance to both reverse transcriptase and protease inhibitors. Continued new infection of this transitory, circulating population of cells even during prolonged, effective HAART most likely reflects ongoing, low-level HIV-1 replication within cellular reservoirs and sanctuary sites in the body.