A V beta TCR repertoire is analyzed for understanding the T-cell population in the immune response. However, the TCR repertoire of the V alpha-V beta pair is difficult to analyze because no suitable analytical method is available. Here, we have applied the single-cell 5'-RACE method for amplifying TCR cDNAs from single T-cells and analyzed the repertoire of V alpha-V beta pairs in human T-cells that responded to a superantigen, SEB. We found that the TCR V beta profile of the SEB-stimulated CD4(+) T-cells was in accordance with the previous reports, that the TCR V alpha profile also exhibited a prominent difference, and that the TCR V alpha-V beta pairs of the SEB-responding T-cells were promiscuous. We have also found a significant dual TCR alpha expression in single T-cells. This is the first report of a comprehensive analysis of the functional repertoire of V alpha-V beta pairs at the single T-cell level. This novel method may contribute to TCR-based immunotherapeutics. (c) 2008 Elsevier Inc. All rights reserved.