A Vbeta TCR repertoire is analyzed for understanding the T-cell population in the immune response. However, the TCR repertoire of the Valpha-Vbeta 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 Valpha-Vbeta pairs in human T-cells that responded to a superantigen, SEB. We found that the TCR Vbeta profile of the SEB-stimulated CD4(+) T-cells was in accordance with the previous reports, that the TCR Valpha profile also exhibited a prominent difference, and that the TCR Valpha-Vbeta pairs of the SEB-responding T-cells were promiscuous. We have also found a significant dual TCRalpha expression in single T-cells. This is the first report of a comprehensive analysis of the functional repertoire of Valpha-Vbeta pairs at the single T-cell level. This novel method may contribute to TCR-based immunotherapeutics.