To better understand the mechanisms of HIV persistence and to further investigate the question on ongoing viral replication in lymph nodes, we characterized HIV proviral populations, their levels of expression, and their sites of host integration in paired lymph node mononuclear cells (LNMC) and peripheral blood mononuclear cells (PBMC) collected after long-term ART and compared to them to the HIV populations prior to ART initiation. Three donors initiated ART in chronic infection and had viremia suppressed for 4-12 years; one donor initiated ART in acute infection and had viremia suppressed for 18 years. Proviral populations and expression were characterized by single-cell analyses and expanded clones were identified using the integration sites assay (ISA). Populations were compared phylogenetically, using a test for panmixia (well-mixed or divergent), by determining the fraction of expressing vs. latent proviruses, determining the levels of expression in single cells with actively-transcribing proviruses, and by comparing the expanded clonal populations. Proviruses in LNMC and PBMC were well mixed and were not significantly divergence from the pre-ART populations. The fraction of proviruses that were latent vs. actively transcribing during ART were not different (5%-8% in the PBMC and 2%-20% in the LNMC) (p=0.4). The levels of expression in actively-infected cells were low in both compartments, typical of suppression of viral replication. The same clonal populations were detected in PBMC and LNMC (p=0.8). These findings are not consistent with continued viral replication during ART in either the peripheral blood or the lymph nodes. Our results also suggest that infected cells migrate freely between the peripheral blood and the lymph nodes and demonstrate that the HIV reservoir is long-lived and proliferating populations of cells that were infected prior to initiating ART.