CD4⁺ T cells have T_h cell function and include two major functional subsets, T_h1 and T_h2. However, there are a restricted number of studies concerning phenotypic classification of human CD4⁺ T cells. Here by using seven- and eight-color flow cytometric analysis, we investigated the function of the subsets classified by four markers, CD27, CD28, CD45RA and CCR7. Five major subsets were identified by using these markers. These subsets showed different patterns of cytokine production after they were stimulated with phorbol myristate acetate and ionomycin. The analyses of cytokine production suggested that CCR7⁺CD45RA⁺CD27⁺CD28⁺, CCR7⁺CD45RA⁻CD27⁺CD28⁺ and CCR7⁻CD45RA⁻CD27⁺CD28⁺ subsets were naive, central memory and effector memory T cells, respectively, whereas CCR7⁻CD45RA⁻CD27⁻CD28⁺ and CCR7⁻CD45RA⁻CD27⁻CD28⁻ subsets included T_h1 and T_h2 cells. The analysis of cytokine production by these subsets stimulated with anti-CD3 and anti-CD28 mAbs or with human cytomegalovirus antigens showed that IFN-γ production was significantly higher in the CCR7⁻CD45RA⁻CD27⁻CD28⁻ subset than in other subsets and that both CCR7⁻CD45RA⁻CD27⁻CD28⁺ and CCR7⁻CD45RA⁻CD27⁻CD28⁻ subsets produced a higher level of IL-4 than did other subsets. Our analyses demonstrated that the CCR7⁻CD45RA⁻CD27⁻CD28⁻ subset predominantly included T_h1 effector cells and that CCR7⁻CD45RA⁻CD27⁻CD28⁺ subsets included T_h1 and T_h2 effector memory/effector cells as well as unclassified cells. The analysis of classification by using these four markers also suggested the differentiation pathway of human CD4⁺ T cells.