Human CMV (HCMV) infection provides an informative model of how long term human CD8+ T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8+ T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8+ T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8+ clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO high cells and the subpopulation of CD45RA high cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8+ T cell memory, we found that in healthy virus carriers highly purified CD28− CD45RA high CCR7− cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8+ T cells are predominantly CD28− CD45RO high CCR7−. During convalescence, an increasing proportion of pp65-specific CD8+ T cells were CD28− CD45RA high CCR7−. We conclude that naive human CD8+ T cells are CD28+ CD45RA high, express CCR7 but not CCR6, and are predominantly CD27+ and L-selectin CD62 ligand-positive. The phenotype CD27+ CD45RA high should not be used to identify naive human CD8+ T cells, because CD27+ CD45RA high cells also contain a significant subpopulation of CD28− CD27+ Ag-experienced expanded clones. Thus CD8+ T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO high and CD28− CD45RA high T cell pools.