High frequencies of EBV-specific CD8+ T cells have been detected during acute EBV infection, yet persistent infection inevitably results. To address this issue, we characterized the phenotype and function of epitope-specific CD8+ T cell populations from presentation with acute through latent infection. Considerable phenotypic and functional heterogeneity within, as well as between, two different epitope-specific populations was observed over time following acute infection. B7 EBV-encoded nuclear Ag (EBNA)-3A-specific CD8+ T cells expressed only CD45RO from acute through latent EBV infection. A2 BMLF-1-specific CD8+ T cells expressed CD45RO during acute infection and either CD45RA or CD45RO during latent EBV infection. This difference in CD45 isoform expression between the two epitope-specific populations did not translate into differences in perforin content, the ability to produce IFN-γ, or the ability to proliferate in response to Ag in vitro. In individuals with latent EBV infection, the frequencies of A2 BMLF-1-or B7 EBNA-3A-specific CD8+ T cells that expressed CD45RA, CD45RO, CD62 ligand, CCR7, and perforin were stable over time. However, the expression of CD62 ligand and CCR7 was significantly higher among EBNA-3A-specific CD8+ T cells than among BMLF-1-specific CD8+ T cells. Further work is necessary to understand how phenotypic and functional differences between EBV epitope-specific CD8+ T cells are related to the biology of the virus and to the equilibrium between the virus and the host during persistent infection.