Treatment of HIV-1-infected individuals with a combination of anti- retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1- infected individuals and for the development of virus eradication strategies.