Phenotypic classification of human CD8(+) T cells using three cell surface markers, CD27, CD28 and CD45RA, was recently suggested to be useful for identification of naive, memory and effector CD8(+) T cells. However, it still remains unclear whether such classification precisely reflects functional classification of CD8(+) T cells. To clarify this, we characterized each CD27CD28CD45RA subset of total and human cytomegalovirus (HCMV)-specific CD8(+) T cells by analyzing the expression of perforin and two chemokine receptors, CCR5 and CCR7, as well as their function. An inverse correlation between perforin and CD27 expression was found in all four CD28CD45RA subsets. Therefore, to achieve a phenotypic classification of CD8(+) T cells that more precisely reflects their function, the CD27(+) subset was divided into CD27(low) and CD27(high) subsets based on the expression level of CD27. Functional and flow cytometric analyses of CD27CD28CD45RA subsets showed that this phenotypic classification reflects functional classification of CD8(+) T cells. HCMV-specific CD8(+) T cells from healthy HCMV-seropositive individuals were predominantly found in effector and memory/effector subsets, indicating that HCMV-specific effector CD8(+) T cells are actively induced by HCMV replication in healthy HCMV carriers. Phenotypic analyses of CD8(+) T cells using this classification will enable the characterization of antigen-specific CD8(+) T cells.