Description: HIV-1 replication is concentrated within CD4 + T cells in B cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (T FH ) within germinal centers (GC) is highly permissive to HIV-1. Whether GC T FH are the major HIV-1 virus–producing cells in vivo has not been established. In this study, we investigated T FH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC T FH (CXCR5 high PD-1 high ) and CXCR5 + programmed cell death-1 (PD-1) low cells were GFP + than non-GC T FH (CXCR5 + PD-1 intermediate ) or extrafollicular (EF) (CXCR5 – ) cells. When sorted prior to spinoculation, however, GC T FH were substantially more permissive than CXCR5 + PD-1 low or EF cells, suggesting that many GC T FH transition to a CXCR5 + PD-1 low phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1–infected individuals without AIDS revealed higher frequencies of HIV-1 RNA + cells in GC than non-GC regions of follicle or EF regions. Superinfection of HIV-1–infected individuals’ lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5 + CD4 + cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5 + subsets harbored 11–66-fold more HIV-1 RNA than CXCR5 – subsets, as determined by RT PCR. Thus, GC T FH are highly permissive to HIV-1, but downregulate PD-1 and, to a lesser extent, CXCR5 during HIV-1 replication. These data further implicate GC T FH as the major HIV-1–producing cells in chronic asymptomatic HIV-1 infection.