Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A* 0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited high-affinity binding in vitro to purified human HLA-A* 0201 molecules. Three of these four peptide epitopes, gD 53–61, gD 70–78, and gD 278–286, significantly stabilized HLA-A* 0201 molecules on T 2 cell lines and are highly conserved among and between HSV-1 and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A* 0201-positive, HSV-1-seropositive, and/or HSV-2-seropositive healthy individuals, the most frequent and robust CD8+ T cell responses, assessed by IFN-γ ELISPOT, CD107a/b cytotoxic degranulation, and tetramer assays, were directed mainly against gD 53–61, gD 70–78, and gD 278–286 epitopes. In addition, CD8+ T cell lines generated by gD 53–61, gD 70–78, and gD 278–286 peptides recognized infected target cells expressing native gD. Lastly, CD8+ T cell responses specific to gD 53–61, gD 70–78, and gD 278–286 epitopes were induced in HLA-A* 0201 transgenic mice following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.