A population of mature CD4-CD8-double-negative T cells that expresses an invariant Valpha24-JalphaQ TCR has been identified in humans; the majority of these cells appear to express Vbeta11. A closely related in variant TCRalpha chain is also expressed by a population of NK1+ murine T cells, but these cells may be either CD4+ or double negative. In this report, multiple CD4+ or double-negative Valpha24+Vbeta11+ T-cell clones were isolated, and only the double-negative clones were found to express the invariant TCRalpha chain. Studies of TCRbeta chains expressed by these cells demonstrate that a subset in some donors use Vbeta genes other than Vbeta11. Characterization of Vbeta11 TCRs in one donor by CDR3-length analysis was also carried out. The results indicate that multiple Vbeta11 TCRs of differing CDR3 lengths may associate with the invariant TCRalpha chain.