Techniques for the quantitation of virus-specific and alloantigen-reactive T cells vary in their measurement of clinically relevant T-cell effector populations, their sensitivity and quantitative accuracy, and the time required to obtain measurable results. We compared frequencies of Epstein-Barr virus (EBV)-specific and major alloantigen-reactive T cells as measured by flow cytometric analysis of responding T cells producing intracellular interferon-gamma (IFN-gamma) and by limiting-dilution analysis (LDA) of cytotoxic T-cell precursors (CTLp) at sequential time points during the generation of EBV-specific T-cell lines. The expansion of EBV-specific T lymphocytes and the depletion of alloreactive T cells in cultures of T cells sensitized with autologous EBV-transformed targets followed similar kinetics when measured by either method. Frequencies of EBV- specific T cells generating intracellular IFN-gamma exceeded by 25- to 90-fold the frequencies of responding CTLp at each stage of expansion, whereas the frequencies of alloreactive T cells generating intracellular IFN-gamma exceeded by 30- to 220-fold those detected by LDA. The assay that quantitated T cells producing IFN-gamma yielded more reproducible and precise results than LDA. Furthermore, frequencies detected by the enumeration of T cells responding to immunodominant EBNA 3a and EBNA 3c peptides by IFN-gamma production or their capacity to bind peptide-HLA tetramers were strikingly similar and represented significant fractions of T cells generating IFN-gamma in response to autologous EBV B lymphoblastoid cell line. Functional analysis of responding viable T cells, fractionated on the basis of their secretion of IFN-gamma, demonstrated that EBV-specific and alloantigen cytotoxic T cells were predominantly or exclusively detected in the CD8(+)IFN-gamma(+) fraction of T cells. Strikingly, the CD4(+)IFN-gamma(+) cell fractions were not cytotoxic against EBV-transformed or allogeneic targets.