HIV-specific CD8⁺ T cells produce antiviral cytokines but are impaired in cytolytic function. This study combines tetramer staining and intracellular cytokine staining to examine the functional heterogeneity of antigen-specific CD8⁺ T cells in HIV infection. HIV-specific CD8⁺ T cells secrete IFN-γ and MIP-1β but not TNF-α, and express significantly lower levels of perforin compared to CMV-specific CD8⁺ T cells. This lack of perforin is linked with persistent CD27 expression, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared to CMV-specific cells from the same donor. HIV-specific CD8⁺ T cells are impaired in cytolytic activity. The study shows that most HIV-specific CD8⁺ T cells are functionally active in cytokine and chemokine secretion, but a subset lacks perforin. The use of specific peptides provides optimal activation for cytokine release. HIV-specific CD8⁺ T cells express low levels of perforin, which is linked to their impaired cytolytic function. The expression of maturation markers differs between HIV-specific and CMV-specific CD8⁺ T cells, with HIV-specific cells showing persistent CD27 expression. These findings suggest that HIV-specific CD8⁺ T cells are at a different stage of maturation compared to CMV-specific cells. The study highlights the importance of functional assays in understanding the role of CD8⁺ T cells in HIV infection.HIV-specific CD8⁺ T cells produce antiviral cytokines but are impaired in cytolytic function. This study combines tetramer staining and intracellular cytokine staining to examine the functional heterogeneity of antigen-specific CD8⁺ T cells in HIV infection. HIV-specific CD8⁺ T cells secrete IFN-γ and MIP-1β but not TNF-α, and express significantly lower levels of perforin compared to CMV-specific CD8⁺ T cells. This lack of perforin is linked with persistent CD27 expression, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared to CMV-specific cells from the same donor. HIV-specific CD8⁺ T cells are impaired in cytolytic activity. The study shows that most HIV-specific CD8⁺ T cells are functionally active in cytokine and chemokine secretion, but a subset lacks perforin. The use of specific peptides provides optimal activation for cytokine release. HIV-specific CD8⁺ T cells express low levels of perforin, which is linked to their impaired cytolytic function. The expression of maturation markers differs between HIV-specific and CMV-specific CD8⁺ T cells, with HIV-specific cells showing persistent CD27 expression. These findings suggest that HIV-specific CD8⁺ T cells are at a different stage of maturation compared to CMV-specific cells. The study highlights the importance of functional assays in understanding the role of CD8⁺ T cells in HIV infection.