Human immunodeficiency virus (HIV) infection is characterized by the massive infiltration of secondary lymphoid organs with activated CD8⁺ T lymphocytes. While converging data indicated that these cells were HIV-specific cytotoxic T lymphocytes (CTLs) responsible for HIV spread limitation, direct evidence was lacking. Here, the presence of HIV-specific effector CTLs was demonstrated directly ex vivo in 15 of 24 microdissected splenic white pulps from an untreated patient and in 1 of 24 tonsil germinal centers from a second patient with incomplete viral suppression following bitherapy. These patients had plasma HIV RNA loads of 5900 and 820 copies per milliliter. The frequencies of HIV-1 DNA⁺ cells in their lymphoid organs were more than 1 in 50 and 1 in 175, respectively. Spliced viral messenger RNA (a marker for ongoing viral replication) was present in most immunocompetent structures tested. Conversely, CTL activity was not found in spleens from 2 patients under highly active antiretroviral therapy, with undetectable plasma viral load. These patients had much lower spleen DNA⁺ cell frequencies (1 in 2700 and 1 in 3800) and no white pulps containing spliced RNA. CTL effector activity as well as spliced viral messenger RNA were both concentrated in the white pulps and germinal centers. This colocalization indicates that viral replication in immunocompetent structures of secondary lymphoid organs triggers anti-HIV effector CTLs to these particular locations, providing clues to target therapeutic intervention.