Little is known regarding the basis for selection of the semi-invariant alpha beta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR-alpha and semi-invariant TCR chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3 beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3 alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi- invariant alpha beta TCR and the unique antigen specificity of NKT cells.