In the present study, we describe the definition of a DRB1*0401 (DR4w4)-specific motif. The strategy used entailed a three-step process: 1) screening a large set of unrelated peptide ligands to identify good MHC binders; 2) truncation analysis of several DR4w4 binding peptides of high affinity to identify the crucial core-binding regions; 3) the use of single amino acid substitutions of the DR4w4-binding peptide hemagglutinin (HA) 307-319 to elucidate the specific residues crucial for binding. The DR4w4 motif is characterized by the presence of a hydrophobic or aromatic (F, W, Y, L, I, V, M) anchor residue in position 1, and a second hydroxyl (S, T) or aliphatic (L, I, V, or M) anchor residue in position 6. Furthermore, positive charges (R, K) are not allowed in positions 4, 7, and 9, and negative charges (D, E) are not allowed in position 9. This motif was present in 92% of good (IC50 < or = 100 nM) DR4w4-binding peptides, but less than 25% of the negative (IC50 > 45 microM) binders, indicating that the presence of the motif is necessary, but not sufficient for good DR4w4 binding capacity. The results of the present study are discussed in relation to previous work defining binding motifs and rules for other DR alleles, illustrating how different DR alleles bind variations on a similar structural theme. Finally, using two different peptide ligands [tetanus toxoid 830-843 and HA 307-319] as model systems, it is demonstrated how the fine allelic specificity of the DR binders can be predictably modulated by introducing subtle changes in the primary amino acid sequence.