Human pegivirus (HPgV; previously called GB virus C/hepatitis G virus) has limited pathogenicity, despite causing persistent infection, and is associated with prolonged survival in human immunodeficiency virus-infected individuals. Although HPgV RNA is found in and produced by T- and B-lymphocytes, the primary permissive cell type(s) are unknown. We quantified HPgV RNA in highly purified CD4(+) and CD8(+) T-cells, including naïve, central memory and effector memory populations, and in B-cells (CD19(+)), NK cells (CD56(+)) and monocytes (CD14(+)) using real-time reverse transcription-PCR. Single-genome sequencing was performed on viruses within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4(+) and CD8(+) T-lymphocytes (nine of nine subjects), B-lymphocytes (seven of ten subjects), NK cells and monocytes (both four of five). HPgV RNA levels were higher in naïve (CD45RA(+)) CD4(+) cells than in central memory and effector memory cells (P<0.01). HPgV sequences were highly conserved among subjects (0.117±0.02 substitutions per site; range 0.58-0.14) and within subjects (0.006±0.003 substitutions per site; range 0.006-0.010). The non-synonymous/synonymous substitution ratio was 0.07, suggesting a low selective pressure. Carboxyfluorescein succinimidyl ester (CFSE)-labelled HPgV RNA-containing particles precipitated by a commercial exosome isolation reagent delivered CSFE to uninfected monocytes, NK cells and T- and B-lymphocytes, and HPgV RNA was transferred to PBMCs with evidence of subsequent virus replication. Thus, HPgV RNA-containing serum particles including microvesicles may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus.