The functional status of virus-specific CD8⁺ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8⁺ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNγ) or tumor necrosis factor-alpha (TNFα). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8⁺ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8⁺ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.