ELISpot and intracellular cytokine staining are replacing the traditional cytolytic ((51)Cr release) assay method in vaccine trials using human immunodeficiency virus (HIV)-1, and it is widely assumed that the number of interferon (IFN)- gamma -secreting T cells is a surrogate for the level of cytolytic activity. Thus, we sought to determine whether the detection of IFN- gamma 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), virus-specific 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 (51)Cr release), and 18 (62.1%) of these 29 demonstrated both IFN- gamma secretion and cytolysis. There was strong correlation between the results of IFN- gamma ELISpot and those of IFN- gamma intracellular cytokine staining ( rho =0.88) and between the results of (51)Cr release and those of intracellular cytokine staining ( rho =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.