To study virus-specific cytotoxic T lymphocyte (CTL) activity at the single cell level, an IFN-γ specific ELISPOT assay was adapted to elucidate the frequency of influenza-specific CTLs together with a standard cytotoxic 51Cr-release assay. Peripheral blood mononuclear cells (PBMCs) from human volunteers were cultured with influenza virus-Infected autologous cells; following 3 or 7 days of culture, T cell subsets were assessed for IFN-γ production by IFN-γ-speclflc ELISPOT and ELISA, while IFN-γmRNA expression was determined by reverse transcrlptase-polymerase chain reaction (RT-PCR). Influenza virus-specific CTL activity was measured in a 4 h 51Cr-release assay. Culture of PBMC with autologous A/Taiwan influenza (H1N1)-infected stimulator cells resulted in IFN-γ spot forming cells (SFCs) at 3 days that increased after 7 days of incubation. Numbers of IFN-γ SFCs directly correlated with levels of secreted IFN-γ and higher levels were seen in supernatants from 7 day cultures. RT-PCR analysis (35 cycles of amplification) showed greater IFN-γ mRNA in T cells isolated from 7 day cultures. Separate allquots of T cells from these cultures were also assessed for virus-specific cytotoxicity and T cells from 7 day (but not from 3 day) cultures induced high 51Cr release. Analysis indicated a significant direct correlation between level of cytotoxicity, number of IFN-γ SFCs, and amount of IFN-γ in culture supematants. Studies with purified T cell subsets showed that elevated IFN-γ SFCs, IFN-γ synthesis, and cytotoxic activity were associated with CD4-CD8+ T cells but not with the CD4+CD8- T cell subset. These results show that increased IFN-γ production, including increased IFN-γ mRNA and IFN-γ SFCs directly correlate with increased antigen-specific, CD8+ T cell mediated cytotoxicity. Thus, assessment of IFN-γ SFCs may provide an alternative and quantitative means for assessment of influenza virus-specific CTL in humans. © 1994 Oxford University Press.