The CD8⁺ CD28⁻ cell population in the blood of HIV‐infected individuals is considerably expanded. Yet the cause of this expansion is not clear. The recent demonstration of identical TCR‐rearranged genes in CD8⁺ CD28⁺ and CD8⁺ CD28⁻ expanded T cells of HIV‐seropositive patients supports the hypothesis that these two subpopulations are phenotypic variants of the same lineage. To further elucidate the precise relationship between them, we measured the fraction of CD28⁺ and CD28⁻ T cell subsets in IFN‐γ‐producing CD8⁺ T cells by intracellular staining and cytofluorometry as a functional test for ex vivo recognition of epitopes derived from HIV‐1, Epstein‐Barr virus (EBV) and influenza virus. HIV‐specific CD8⁺ T cells were predominantly CD28⁻ in all the eight HIV‐seropositive subjects tested. In contrast, the anti‐EBV and anti‐influenza CD8⁺ T cells were mainly CD28⁺ in these patients as well as in HIV‐seronegative individuals. This supports the notion that the CD8⁺ CD28⁻ hyperlymphocytosis observed in HIV infection is due mainly to chronic activation and differentiation of HIV‐specific memory CD8⁺ CD28⁺ T cells into terminally differentiated CD8⁺ CD28⁻ lymphocytes, because of intense HIV‐1 replication and without any important bystander activation. This clarification of the mechanisms underlying the CD8⁺ CD28⁻ expansion in HIV‐induced pathogenesis may have important therapeutic implications.