CD1d-restricted natural killer (NK) T cells reactive with the glycolipid α-galactosylceramide (α-GalCer) are a distinct lymphocyte sublineage. They express an invariant Vα14-Jα18 T cell receptor (TcR), but the role of the β chain has been controversial. Here, we have used CD1d tetramers to identify and isolate NK T cells based on their antigen specificity. In mice lacking germline Vβ8, most of the α-GalCer-reactive T cells express either Vβ2 or Vβ7, strong Vβ selection being revealed by the lack of an increase in other Vβ regions. By contrast to the selection for complementarity determining region (CDR) 3β sequences in some anti-peptide responses, α-GalCer-reactive T cells have polyclonal CDR3β sequences. There is little CDR3β sequence redundancy between organs or individual mice, and, surprisingly, there also is no evidence for organ-specific CDR3β sequence motifs. These data argue against a T cell receptor-mediated self-reactivity for tissue-specific CD1d-bound ligands. Each NKT clone is represented by only 5–10 cells. This clone size is similar to naive conventional T cells, and much lower than that reported for memory T cells, although NK T cells have an activated/memory phenotype.