CD8⁺ T cells play a crucial role in the control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. Recent data suggest that HIV-1-specific CD8⁺ T cell subsets may differ in their ability to exert these effector functions. Here, we directly compared the cytokine secretion patterns and cytotoxic capacity of HIV-1-specific CD8⁺ T cells, using a flow-cytometric cytotoxicity assay based on caspase-3 activation in dying target cells. These experiments revealed considerable intraindividual and interindividual differences among epitope-specific T-cell effector functions: while the frequency of HIV-1-specific CD8⁺ T cells secreting interferon-γ but no tumor necrosis factor-α (TNF-α) following antigenic stimulation was only weakly correlated to their cytotoxic activity (R = 0.05, P = .57), a subset of CD8⁺ T cells secreting both inter-feron-γ and TNF-α was substantially more strongly associated with cytotoxicity (R = 0.67, P < .001). This subset of CD8⁺ T cells also exhibited stronger intracellular perforin expression and more pronounced direct ex vivo HIV-1-specific cytoxicity than CD8⁺ T cells secreting solely interferon-γ following sorting of these subpopulations according to their cytokine profile. These results suggest that HIV-1-specific cytotoxicity of CD8⁺ T cells is preferentially mediated by a subset of CD8⁺ T cells secreting both interferon-γ and TNF-α. (Blood. 2004;104:487-494)