The observation that HIV-1 replication is concentrated in lymphoid follicles (F) compared to extrafollicular (EF) lymphoid tissue is not fully understood. The purpose of this study was to quantify HIV-1 replication in these compartments and evaluate the hypothesis that heightened replication in F occurs because of diminished antiretroviral mechanisms. In situ hybridization for HIV-1 RNA and immunohistochemical staining for CD4, CD8, CD20, and multiple antiretroviral proteins was performed in lymph nodes from 15 HIV-1-infected individuals who were not receiving antiretroviral therapy. A median of 70% of virus-producing cells were detected in F, defined morphologically by CD20 staining, although only a minority of tissue (median, 19%) consisted of F. Frequencies of virus-producing cells were higher in F (median, 1.35 cells/mm2) compared to EF (median, 0.35 cells/mm2; p < 0.0001). A CD4+ cell in F had a median 31-fold greater likelihood of harboring HIV-1 RNA than a CD4+ cell in EF (p = 0.0006). The most highly expressed antiretroviral proteins were alpha-defensins 1, 2, and 3 (median, 4.6% tissue staining), RANTES (median, 728.4 cells/mm2), and granzyme A (median, 412.6 cells/mm2). Less alpha-defensins (p = 0.0127), RANTES (p = 0.0007), granzyme A (p = 0.0018), MIP-1alpha (p = 0.0054), interferon (IFN)-alpha (p = 0.0186), and CD8 (p < 0.0001) was expressed in F compared to EF; amounts in F ranged from 0.15 to 0.50 of those in EF. Expression of IFN-gamma (median, 3.1 cells/mm2) and perforin (median, 4.0 cells/mm2) was low and not significantly different in F and EF. Deficiencies of multiple antiretroviral proteins within F may contribute to heightened replication at that site.