Assessment of the quality and the breadth of antigen (Ag)-specific memory T cells in human samples is of paramount importance to elucidate the pathogenesis and to develop new treatments in various diseases. T-cell receptor (TCR) signal strength, primarily controlled by TCR affinity, affects many fundamental aspects of T-cell biology; however, no current assays for detection of Ag-specific CD8(+) T cells can assess their TCR signal strength in human samples. Here, we provide evidence that interferon regulatory factor 4 (IRF4), a transcription factor rapidly upregulated in correlation with TCR signal strength, permits the assessment of the TCR signal strength of Ag-specific CD8(+) T cells in human peripheral blood mononuclear cells (PBMCs). Coexpression of IRF4 and CD137 sensitively detected peptide-specific CD8(+) T cells with extremely low background in PBMCs stimulated for 18 hours with MHC class I peptides. Our assay revealed that human memory CD8(+) T cells with high-affinity TCRs have an intrinsic ability to highly express CD25. Furthermore, HIV-specific CD8(+) T cells in chronic HIV(+) subjects were found to display primarily low-affinity TCRs with low CD25 expression capacity. Impairment in the functions of HIV-specific CD8(+) T cells might be associated with their suboptimal TCR signals, as well as impaired responsiveness to interleukin-2.