To discuss the role of CD4+ T cells with active Human immunodeficiency virus (HIV), meaning infected cells with transcriptional and/or translational viral activity during antiretroviral therapy (ART), focusing on new technologies for its detection, potential cell markers for its characterization, and evidences on the contribution of the active HIV reservoir to long-term viral persistence. HIV-infected cells expressing viral ribonucleic acid are systematically detected in subjects on long-term ART. In recent years, powerful new tools have provided significant insights into the nature, quantification, and identification of cells with active HIV, including the identification of new cell markers, and the presence of viral activity in specific cell populations located in different cellular and anatomical compartments. Moreover, studies on viral sequence integrity have identified cell clones with intact viral genomes and active viral transcription that could potentially persist for years. Together, new investigations support the notion that the active reservoir could represent a relevant fraction of long-term infected cells, and therefore, the study of its cell sources and mechanisms of maintenance could represent a significant advance in our understanding of viral persistence and the development of new curative strategies. The presence of HIV-infected cells with viral expression during ART has been traditionally overlooked for years. Based on recent investigations, this active viral reservoir could play an important role in HIV persistence.