ELISpot and intracellular cytokine staining are replacing the traditional cytolytic (₅₁Cr release) assay method in vaccine trials using human immunodeficiency virus (HIV)-1, and it is widely assumed that the number of interferon (IFN)-γ-secreting T cells is a surrogate for the level of cytolytic activity. Thus, we sought to determine whether the detection of IFN-γ in CD8⁺ T cells correlates with cytolytic ability in vitro. In 29 (69.0%) of 42 HIV-1-seronegative immunocompetent individuals (22 unvaccinated and 20 vaccinated), virusspecific T cell responses recognizing cytomegalovirus, Epstein- Barr virus, and influenza and HIV-1 Gag epitopes were detected by at least 1 assay method (ELISpot, intracellular cytokine staining, and/or ⁵¹Cr release), and 18 (62.1%) of these 29 demonstrated both IFN-g secretion and cytolysis. There was strong correlation between the results of IFN-γ ELISpot and those of IFN-γ intracellular cytokine staining (ρ = 0.88) and between the results of ⁵¹Cr release and those of 0.88 intracellular cytokine staining (ρ = 0.81 ); although the correlation is not absolute, intracellular cytokine staining can be used—and is superior to ELISpot—as a surrogate for cytolytic assays.