Natural killer T cells express a conserved, semi-invariant αβ T cell receptor that has specificity for self glycosphingolipids and microbial cell wall α-glycuronosylceramide antigens presented by CD1d molecules. Here we report the crystal structure of CD1d in complex with a short-chain synthetic variant of α-galactosylceramide at a resolution of 2.2 Å. This structure elucidates the basis for the high specificity of these microbial ligands and explains the restriction of the α-linkage as a unique pathogen-specific pattern-recognition motif. Comparison of the binding of altered lipid ligands to CD1d and T cell receptors suggested that the differential T helper type 1–like and T helper type 2–like properties of natural killer T cells may originate largely from differences in their 'loading' in different cell types and hence in their tissue distribution in vivo.