Human immunodeficiency virus type 1 (HIV-1) RNA that persists in the lymphoid tissue of patients despite treatment with highly active antiretroviral therapy (HAART) may represent extracellular virions or intracellular RNAs residing within HIV-infected cells. To further characterize residual viral transcription, tonsil biopsy specimens from patients receiving long-term HAART, untreated patients, and patients undergoing 2 weeks of structured treatment interruption were analyzed by polymerase chain reaction quantification of virion-encapsidated RNA, intracellular unspliced HIV RNA (HIV UsRNA), multiply spliced HIV RNA encoding tat and rev (HIV MsRNA), and HIV DNA. Tonsil biopsy specimens from viremic patients harbored high amounts of virions, which primarily stemmed from local production, as indicated by a strong correlation of extracellular tonsillar RNA with intracellular HIV-1 nucleic acid levels but not with plasma viremia, and as shown by phylogenetic analysis of clonal env sequences from lymphoid tissue and plasma. In patients receiving HAART, intracellular HIV UsRNA persisted at significantly decreased levels, whereas HIV MsRNA and lymphoid virion levels were depleted. Thus, residual lymphoid HIV-1 RNA in patients receiving HAART indicates attenuated viral transcription in HIV-1–infected cells that lack virion production