Cells that actively transcribe HIV-1 have been defined as the "active viral reservoir" in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4 + T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4 + T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4 + T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation. Persons infected with HIV-1 contain several cellular viral reservoirs that preclude the complete eradication of the viral infection. Using a novel methodology, we identified effector memory CD4 + T cells, immune cells preferentially located in inflamed tissues with potent activity against pathogens, as the main cells encompassing the transcriptionally active HIV-1 reservoir in patients on antiretroviral therapy. Importantly, the identification of such cells provides us with an important target for new therapies designed to target the hidden virus and thus to eliminate the virus from the human body. In addition, because of its ability to identify cells forming part of the viral reservoir, the assay used in this study represents an important new tool in the field of HIV pathogenesis and viral persistence.