Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA− CD8+ T cells with different CD28/CD27 phenotypes showed that CD28+ CD45RA− CD8+ and CD27+ CD45RA− CD8+ T cells have characteristics of memory T cells, whereas CD28− CD45RA− CD8+ and CD27− CD45RA− CD8+ T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8+ T cells into memory/effector CD8+ T cells has not been completely clarified. We investigated this differentiation pathway using EBV-and human CMV (HCMV)-specific CD8+ T cells. Three subsets of CD45RA− CD8+ T cells were observed in both total CD8+ T cells and EBV-or HCMV-specific CD8+ T cells: CD27+ CD28+, CD27+ CD28−, and CD27− CD28−. A significant number of the CD27− CD28+ subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV-or HCMV-specific CD8+ T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27+ CD28+ CD45RA−→ CD27+ CD28− CD45RA−→ CD27− CD28− CD45RA−. This was supported by the observation that the frequency of CCR5+ cells and CCR7+ cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27+ CD28+ memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5− CCR7+, CCR5+ CCR7+, and CCR5+ CCR7−. These findings suggested the following differentiation pathway: CD27+ CD28+ CD45RA−(CCR5− CCR7+→ CCR5+ CCR7+→ CCR5+ CCR7−)→ CD27+ CD28− CD45RA−→ CD27− CD28− CD45RA−. The presence of a CD27− CD28+ subset with a CCR5+ CCR7− phenotype implies a specialized role for this subset in the differentiation of CD8+ T cells.