Solution at 2.5-Å resolution of the three-dimensional structure of H-2Ldwith a single nine-residue peptide provides a structural basis for understanding its unique interaction with beta-2 microglobulin (β2m) and peptide. Consistent with the biological data that show an unusually weak association of Ldwith β2m, a novel orientation of the α1/α2 domains of Ldrelative to β2m results in a dearth of productive contacts compared with other class I proteins. Characteristics of the Ldantigen-binding cleft determine the unique motif of peptides that it binds. Ldhas no central anchor residue due to the presence of several bulky side chains in its mid-cleft region. Also, its cleft is significantly more hydrophobic than that of the other class I molecules for which structures are known, resulting in many fewer H-bonds between peptide and cleft residues. The choice of Pro as a consensus anchor at peptide position 2 appears to be related to the hydrophobicity of the B pocket, and to the unique occurrence of Ile (which mirrors Pro in its inability to form H-bonds) at position 63 on the edge of this pocket. Thus, the paucity of stabilizing H-bonds combined with poor complementarity between peptide postion 2 Pro and the B pocket contribute to the weak association between Ldand its peptide antigen. The unique structural interactions of Ldwith β2m and peptide could make Ldmore suited than other classical class I molecules to play a role in alternative pathways of antigen presentation.