Unspliced HIV-RNA (HIV-UsRNA) associated with peripheral blood mononuclear cells (PBMCs) persists in patients on potent antiretroviral therapy even in the absence of detectable plasma HIV-RNA. To further characterize such residual HIV-RNA, cell-associated virion-encapsidated HIV and intracellular unspliced HIV-RNA were differentiated and monitored using a novel highly sensitive method. In addition, expression of HIV-mRNA encoding tat and rev was assessed. PBMCs of patients with unsuppressed plasma viraemia harboured an extracellular fraction of HIV-UsRNA, which correlated highly with intracellular HIV-RNA levels. Thus, extracellular PBMC-associated HIV-RNA may, to a significant extent, reflect nascent virions attached to productively infected cells. Upon treatment with potent antiretroviral therapy resulting in plasma viraemia <50 copies/ml, expression of cell-associated viral particles was hardly discernible in PBMCs but transcription of unspliced HIV-RNA persisted. Given the virtual absence of rev-mRNA, translation of residual HIV-UsRNA was probably precluded by retention of these transcripts in the nucleus. As shown by limiting dilution analysis, HIV-1 infected cells with such a repressed viral transcription pattern were observed at high frequencies in PBMC from untreated patients.