Major histocompatibility class I–peptide tetramer technology and simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys were used to clarify the distribution of acquired immunodeficiency syndrome virus-specific cytotoxic T lymphocytes (CTL) in secondary lymphoid organs and to assess the relationship between these CTL and the extent of viral replication in the various anatomic compartments. SIVmac Gag epitope-specific CD8⁺ T cells were evaluated in the spleen, bone marrow, tonsils, thymus, and 5 different lymph node compartments of 4 SIVmac-infected rhesus monkeys. The average percentage of CD8⁺ T lymphocytes that bound this tetramer in all the different lymph node compartments was similar to that in peripheral blood lymphocytes in individual monkeys. The percentage of CD8⁺ T cells that bound the tetramer in the thymus was uniformly low in the monkeys. However, the percentage of CD8⁺ T cells that bound the tetramer in bone marrow and spleen was consistently higher than that seen in lymph nodes and peripheral blood. The phenotypic profile of the tetramer-binding CD8⁺ T lymphocytes in the different lymphoid compartments was similar, showing a high expression of activation-associated adhesion molecules and a low level expression of naive T-cell–associated molecules. Surprisingly, no correlation was evident between the percentage of tetramer-binding CD8⁺ T lymphocytes and the magnitude of the cell-associated SIV RNA level in each lymphoid compartment of individual monkeys. These studies suggest that a dynamic process of trafficking may obscure the tendency of CTL to localize in particular regional lymph nodes or that some lymphoid organs may provide milieus that are particularly conducive to CTL expansion.