Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIVRNA/Gag method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5-1 Gagpol mRNA+/Gag protein+ cells per million CD4 T cells. Uniquely, the HIVRNA/Gag assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.