Human NKRP-1A (CD161)+ T cells include members of a family of CD4+ or CD4-/CD8- lymphocytes that utilize an invariant alpha chain in the T-cell receptor (TCR). The alpha chain consists of the Valpha24 segment joined to Jalpha18 (JalphaQ) (TCRAV24/AJ18). These families of T cells rapidly produce both interleukin-4 and interferon-gamma upon TCR cross-linking, and are restricted by CD1d. To determine the spectrum of allowable V/J rearrangements in the Valpha24+ CD4-/CD8- family, TCR Valpha24 chain transcripts derived from the total CD4-/CD8- population in peripheral blood mononuclear cells were sequenced. A second invariant rearrangement, Valpha24Jalpha45, was found in two donors. In addition, a subset of 15 clones with single amino acid substitutions in the CDR3 were identified and used to define CD1d restriction. All 15 variant clones were indistinguishable from invariant clones on the basis of surface phenotype and response to CD3 cross-linking. However, CD1d was the restriction element only for those clones with the conservative substitution of threonine or asparagine for serine at the V/J junction. Thus, the family of human CD161+ T cells can be extended to include a subset of Valpha24-JalphaQ rearrangements with a single amino acid substitution that defines a Valpha24 CDR3 residue critical for CD1d restriction.