Residual viremia can be detected in most HIV-1-infected patients on antiretroviral therapy despite suppression of plasma RNA to <50 copies per ml, but the source and duration of this viremia is currently unknown. Therefore, we analyzed longitudinal plasma samples from 40 patients enrolled in the Abbott M97-720 trial at baseline (pretherapy) and weeks 60 to 384 by using an HIV-1 RNA assay with single-copy sensitivity. All patients were on therapy (lopinavir/ritonavir, stavudine, and lamivudine) with plasma HIV RNA <50 copies per ml by week 96 of the study and thereafter. Single-copy assay results revealed that 77% of the patient samples had detectable low-level viremia (>/=1 copy per ml), and all patients had at least one sample with detectable viremia. A nonlinear mixed effects model revealed a biphasic decline in plasma RNA levels occurring over weeks 60 to 384: an initial phase of decay with a half-life of 39 weeks and a subsequent phase with no perceptible decay. The level of pretherapy viremia extrapolated for each phase of decay was significantly correlated with total baseline viremia for each patient (R(2) = 0.27, P = 0.001 and R(2) = 0.19, P < 0.005, respectively), supporting a biological link between the extent of overall baseline viral infection and the infection of long-lived reservoirs. These data suggest that low-level persistent viremia appears to arise from at least two cell compartments, one in which viral production decays over time and a second in which viral production remains stable for at least 7 years.