Highly active antiretroviral therapy can decrease plasma HIV-1 levels to below the limit of detection. However, HIV-1 persists in latently infected resting-memory CD4+ T-cells carrying an integrated copy of the viral genome. The pool of latently infected cells is extremely stable and represents a major barrier to HIV-1 eradication. Identification and characterization of this reservoir required the development of methods for purifying resting CD4+ T-cells from HIV-1-infected individuals, activating the cells to induce virus production, and detecting and quantitating cells capable of releasing infectious virus. The development of an enhanced viral culture assay to quantitate the number of latently infected cells carrying replication competent virus is described here.