HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic and progressive disorder caused by the human T-lymphotropic virus type 1 (HTLV-1). In HTLV-1 infection, a strong cytotoxic T cell (CTL) response is mounted against the immunodominant protein Tax. Previous studies carried out by our group reported that increased IFN-gamma enzyme-linked immunospot (ELISPOT) responses against the region spanning aminoacids 161 to 233 of the Tax protein were associated with HAM/TSP and increased HTLV-1 proviral load (PVL). An exploratory study was conducted on 16 subjects with HAM/TSP, 13 asymptomatic carriers (AC), and 10 HTLV-1-seronegative controls (SC) to map the HAM/TSP associated CTL epitopes within Tax region 161-233. The PVL of the infected subjects was determined and the specific CTL response was evaluated with a 6 h incubation IFN-gamma ELISPOT assay using peripheral blood mononuclear cells (PBMC) stimulated with 16 individual overlapping peptides covering the Tax region 161-233. Other pro-inflammatory and Th1/Th2 cytokines were also quantified in the supernatants by a flow cytometry multiplex assay. In addition, a set of human leukocyte antigen (HLA) class I alleles that bind with high affinity to the CTL epitopes of interest was determined using computational tools. Univariate analyses identified an association between ELISPOT responses to two new CTL epitopes, Tax 173-185, and Tax 181-193, and the presence of HAM/TSP as well as an increased PVL. The HLA-A*6801 , which is predicted to bind to Tax 181-193 peptide, was over-presented in the HAM/TSP patients tested.