The existence of a reservoir of resting CD4+ T cells harboring latent replication-competent HIV has been demonstrated in patients on prolonged highly active antiretroviral therapy (HAART). Latently infected tissue macrophages may constitute a second HIV reservoir. The pool of these cells may be maintained by incoming infected monocytes from blood and/or by in situ viral replication. In this study, the presence of infectious HIV was investigated in highly purified monocytes from 5 patients receiving HAART with undetectable plasma viral load for up to 16 months. HIV was detected in freshly isolated monocytes and recovered following Staphylococcus aureus Cowan strain 1 (SAC) or lipopolysaccharide (LPS) activation. No new drug resistance-associated mutation was found in monocyte-associated HIV. These results demonstrate the long-term persistence of infectious virus in cells of the monocyte-macrophage lineage in patients receiving HAART. These cells are capable of releasing infectious virus under appropriate stimulations.