Human herpesvirus 8 (HHV-8; Kaposi sarcoma–associated herpesvirus)–specific cytotoxic T-lymphocyte (CTL) and interferon-γ (IFN-γ) responses to proteins produced during the lytic cycle of HHV-8 replication are mediated by HLA class I–restricted, CD8⁺ T cells. We have characterized the fine specificity of the CD8⁺ T-cell response to 25 peptides derived from 5 HHV-8 lytic cycle proteins based on a prediction model for HLA A*0201 binding motifs. One of the 25 HLA A*0201 peptides derived from the glycoprotein B (gB) homolog of Epstein-Barr virus (gB_492-500; LMWYELSKI; single-letter amino acid codes) bound to HLA A*0201 and stimulated IFN-γ responses in CD8⁺ T cells from HHV-8⁺, HLA A*0201 persons, but not HHV-8–seronegative or non–HLA A*0201 persons. The peptide also induced IFN-γ and CTL reactivity to naturally processed gB protein. The peptide was a major immunogenic epitope of HHV-8 as indicated by induction of IFN-γ responses in peripheral blood mononuclear cells from 5 of 5 HHV-8 seropositive, HLA A*0201 persons when gB_492-500 was presented by autologous dendritic cells. T-cell reactivity to gB_492-500 was not related to detectable HHV-8 DNA in the blood. These data show that CD8⁺ T cells recognize an HLA A*0201–restricted epitope for HHV-8 lytic cycle protein gB, particularly when presented by dendritic cells. This epitope may be important in control of HHV-8 infection by CD8⁺ T cells.