Percentages of activated T cells correlate with HIV-1 disease progression, but the underlying mechanisms are not fully understood. We hypothesized that HLA-DR⁺ CD38⁺ (DR⁺ 38⁺) CD4⁺ T cells produce the majority of HIV-1 due to elevated expression of CCR5 and CXCR4. In phytohemagglutinin (PHA)-stimulated CD8-depleted peripheral blood mononuclear cells (PBMC) infected with HIV-1 green fluorescent protein (GFP) reporter viruses, DR⁻ 38⁺ T cells constituted the majority of CCR5 (R5)-tropic (median, 62%) and CXCR4 (X4)-tropic HIV-1-producing cells (median, 61%), although cell surface CCR5 and CXCR4 were not elevated in this subset of cells. In lymph nodes from untreated individuals infected with R5-tropic HIV-1, percentages of CCR5⁺ cells were elevated in DR⁺ 38⁺ CD4⁺ T cells (median, 36.4%) compared to other CD4⁺ T-cell subsets (median values of 5.7% for DR⁻ 38⁻ cells, 19.4% for DR⁺ 38⁻ cells, and 7.6% for DR⁻ 38⁺ cells; n = 18; P < 0.001). In sorted CD8⁻ lymph node T cells, median HIV-1 RNA copies/10⁵ cells was higher for DR⁺ 38⁺ cells (1.8 × 10⁶) than for DR⁻ 38⁻ (0.007 × 10⁶), DR⁻ 38⁺ (0.064 × 10⁶), and DR⁺ 38⁻ (0.18 × 10⁶) subsets (n = 8; P < 0.001 for all). After adjusting for percentages of subsets, a median of 87% of viral RNA was harbored by DR⁺ 38⁺ cells. Percentages of CCR5⁺ CD4⁺ T cells and concentrations of CCR5 molecules among subsets predicted HIV-1 RNA levels among CD8⁻ DR/38 subsets (P < 0.001 for both). Median HIV-1 DNA copies/10⁵ cells was higher in DR⁺ 38⁺ cells (5,360) than in the DR⁻ 38⁻ (906), DR⁻ 38⁺ (814), and DR⁺ 38⁻ (1,984) subsets (n = 7; P ≤ 0.031). Thus, DR⁺ 38⁺ CD4⁺ T cells in lymph nodes have elevated CCR5 expression, are highly susceptible to infection with R5-tropic virus, and produce the majority of R5-tropic HIV-1. PBMC assays failed to recapitulate in vivo findings, suggesting limited utility. Strategies to reduce numbers of DR⁺ 38⁺ CD4⁺ T cells may substantially inhibit HIV-1 replication.