The enumeration of blood CD4+ (single-positive) T cells by flow cytometry is subject to errors such as counting CD4+ monocytes or CD4+CD8+ (double-positive) T cells as CD4+ T cells. Relatively accurate count can be obtained when CD4+ T cells are defined as CD3+CD4+CD8- mononuclear cells (MNCs), using 3-color flow cytometry. However, using this approach, further classification into CD4+ T cell subsets requires expensive 4-color flow cytometry. In an attempt to enumerate CD4+ T cells using only 2 colors, we have defined CD4+ T cells as MNCs expressing CD4 and not expressing the markers of other MNCs (CD8, CD13, CD14, and CD16) and compared the results to those obtained with the 3-color method in which CD4+ T cells were defined as CD3+CD4+CD8- MNCs. Both methods produced similar results. An analogous approach was undertaken to enumerate CD8+ T cells as MNCs expressing CD8 and not expressing the markers of other MNCs. However, when compared with the percents obtained by the 3-color method in which CD8+ T cells were defmed as CD3+CD4-CD8+ MNCs, the 2-color method overestimated the percent of CD8+ T cells. This was likely due to counting CD8low null cells as CD8+ T cells. When the percentages of CD8high T cells were evaluated, both methods produced similar results. We conclude that the 2-color method is suitable for the enumeration of CD4+ T cells and CD8high T cells and reserves the third color for further enumeration of CD4+ or CD8high T cell subsets.