The significance of distinct classes of HIV-1 nucleic acids as correlates of recent HIV-1 replication was assessed in peripheral blood mononuclear cells (PBMC) obtained from 14 patients during 2 weeks of structured interruption of antiretroviral therapy (STI) and 2 weeks of resuming therapy. Levels of HIV RNA in plasma (HIV-RNAplasma) and of unspliced cell-associated HIV-1 RNA (HIV-UsRNAPBMC) were significantly increased as a result of STI, whereas no significant shifts in the levels of 2-LTR episomal HIV-1 DNA (2-LTR circles) and total late HIV-1 reverse transcripts (late-DNA) were observed. Thus, limited viral replication had occurred, which had no effect on the pool size of infected cells in the periphery. Levels of 2-LTR circles did not reflect rapid changes in HIV-1 replication. In contrast, expression of HIV-UsRNAPBMC increased during STI and consequently provides a more sensitive, albeit not absolute cellular marker of ongoing HIV-1 replication.