Peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type I (HTLV-I)–associated myelopathy/tropical spastic paraparesis (HAM/TSP) proliferate spontaneously in vitro. This spontaneous lymphoproliferation (SP) is one of the immunologic hallmarks of HAM/TSP and is considered to be an important factor related to the pathogenesis of HAM/TSP. However, the cell populations involved in this phenomenon have not yet been definitively identified. To address this issue, the study directly evaluated proliferating cell subsets in SP with a flow cytometric method using bromodeoxyuridine and Ki-67. Although both CD4⁺ and CD8⁺ T cells proliferated spontaneously, the percentage of proliferating CD8⁺ T cells was 2 to 5 times higher than that of CD4⁺ T cells. In addition, more than 40% of HTLV-I Tax11-19–specific CD8⁺T cells as detected by an HLA-A*0201/Tax11-19 tetramer proliferated in culture. In spite of this expansion of HTLV-I–specific CD8⁺ T cells, HTLV-I proviral load did not decrease. This finding will help elucidate the dynamics of in vivo virus-host immunologic interactions that permit the coexistence of high HTLV-I–specific CD8⁺ cytotoxic T-lymphocyte responses and high HTLV-I proviral load in HAM/TSP.