The frequency of the HLA-DR4-DQ4 haplotype (DRB1(*)0405-DQA1(*)0302-DQB1(*)0401) is significantly increased in Japanese patients with rheumatoid arthritis (RA) and DRB1(*)0405-binding peptide motifs were identified in our previous studies. To clarify the DQ4-binding peptide motifs, the primary structure of DQ4-binding peptides was determined by affinity-based selection of a phage random peptide library. Analog peptides of a high-affinity DQ4 binder revealed that two major anchors (VxxxxxxxR; where x is any amino acid) play an essential role in binding to DQ4. The affinity of synthetic VAAAAAAAR-based analog peptides showed that substituting V to W, G, L, I, M, P, F, Y or A and R to H, M, L, I or V allows binding. The involvement of the ninth residue of the peptides, especially Arg, was critical for high-affinity binding. In comparison with other class II-binding peptide motifs reported to date, peptide motifs for DQ4 were unique, in that Gly and Pro are allowed as low-affinity N-terminal anchors. Interestingly, 94 putative DQ4-binding motifs were detected in the human type II collagen molecule, since it is composed of (Gly-X1-X2)n and is rich in R and P at positions X2. However, no significant differences were observed between the affinities of the collagen-derived peptides with DR or DQ molecules of RA-susceptible DR4-DQ4 and with those of non-susceptible DR4-DQ8 (DRB1(*)0406-DQA1(*)0301-DQB1(*)0302) haplotypes, indicating that the susceptibility to RA is not a simple immune response gene phenomenon specific to collagen. The immunogenetic implications of the unique peptide motifs for DQ are discussed.