cells. We found that infection of cytokine-treated resting CD4؉ T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yielded de novo virus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression and de novo virus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.