To characterize newly arising replication of human immunodeficiency virus (HIV) type 1 in vivo at the cellular level, distinct viral RNA species in peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients were monitored during 2 weeks of structured treatment interruption (STI). HIV-1 RNA encoding tat/rev and PBMC-associated virions were almost completely depleted during antiretroviral therapy and emerged simultaneously after 2 weeks of STI, thus specifically reflecting productive viral infection at the cellular level. The magnitude of these correlates of reappearing cellular viral replication was predicted by during-therapy levels of nef transcripts in PBMCs. Significant rebound of plasma viremia, representing the progeny of a broader range of anatomical compartments, preceded and predicted productive infection in PBMCs. Thus, cellular viral rebound in PBMCs likely was primed before STI by the expression of nef in HIV-1-infected PBMCs that lacked virion production and was subsequently triggered by the plasma viremia that preceded the recurrence of productively infected PBMCs.