The peptide motif of the HLA-DR53 (DRB4(*)0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada's syndrome, was determined by peptide binding assay using human L plastin p581 - 595 peptide and its substituted analogues. L plastin p581 - 595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1(*)0901/DRB4(*)0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules.