CD1d molecules recognize glycolipid antigens with straight chain fatty acid moieties. Although most of the residues in the CD1d binding groove are hydrophobic, some of the amino acids can form hydrogen bonds. Consequently, we have designed ω-hydroxy fatty acid-containing glycolipid derivatives of the prototypical CD1d ligand α-GalCer. The potency of the ω-hydroxy analogues of the proper length is comparable to that of α-GalCer. We propose, based on the biological results and molecular modeling studies, that a hydrogen bonding interaction is involved between the ω-hydroxy group and a polar amino acid residue in the hydrophobic binding groove.