Oligonucleotides encoding peptides known to bind to HLA-DR1 molecules have been inserted into the gene III of filamentous M13 phages. DR1 molecules purified from human lymphoblastoid cell lines could specifically bind to these peptide sequences expressed on the phage surface. A M13 phage peptide library was next constructed and screened with DR1 molecules. After four rounds of selection, more than 80% of the phages were able to bind to DR1. Competition experiments with both isolated phages and corresponding synthetic peptides showed that the binding was specific. Sequence analysis of the peptide encoding region of 60 phages binding to DR1 molecules and comparison with phages of the original library revealed two potential anchor positions. The first was an aromatic residue (Tyr, Phe, or Trp) at the NH2 terminus of the peptide sequences, and the second was located three residues downstream and consisted of Met or Leu. In addition, the negatively charged amino acids Asp and Glu were mostly excluded from the DR1 binding sequences, and the small amino acid residues Gly and Ala were enriched at position 6. As for DR1, this approach should enable one to easily determine the binding motifs of other MHC class II alleles and isotypes. Furthermore, it could have interesting applications in the design of major histocompatibility complex-specific antagonists.