Multi‐color flow cytometric analysis on human CD8⁺ T cell subsets revealed that CXCR4 is predominantly expressed on CD8⁺ T cells with the naive CD27⁺CD28⁺CD45RA⁺ phenotype, and is down‐regulated during differentiation into those with an effector phenotype. The down‐regulation of CXCR4 expression during peripheral differentiation was supported by the fact that the expression of CXCR4 on CD8⁺ T cells was negatively correlated with that of perforin. The analysis of CCR5, CCR7, and CXCR4 co‐expression further showed that CD8⁺ T cells expressing a high level of CXCR4 are CCR7⁺CCR5⁻ naive or central memory subsets, and those expressing a low level of CXCR4 were included in the CCR7⁻CCR5^+/− memory/effector and effector subsets. Epstein Barr virus‐specific CD8⁺ T cells, which mostly express the memory phenotype, expressed CXCR4, while human cytomegalovirus‐specific CD8⁺ T cells, which mostly express the effector phenotype, partially expressed this receptor, showing that the expression of CXCR4 is also down‐regulated during differentiation of viral antigen‐specific CD8⁺ T cells. The classification of human CD8⁺ T cells based on the expression of these chemokine receptors should prove useful for studies that clarify the differentiation of human CD8⁺ T cells.