The third hypervariable region (HV3) of HLA-DRB1*0401 helps the development of severe rheumatoid arthritis by an unknown mechanism. To test whether the third hypervariable region of HLA-DRB1*0401 may shape the T cell repertoire, we studied proliferative responses to peptides encompassing the third hypervariable region of seven HLA-DRB1 alleles in normal subjects and patients with rheumatoid arthritis. We found that, in general, there is no tolerance to peptides from the third hypervariable region of self-HLA-DRB1 alleles. However, a peptide from the third hypervariable region of DRB1*0401 (DRB1*0401 HV3 peptide) is tolerated in people who express HLA-DRB1*0401. Indeed, PBMCs from people who express DRB1*0401 do not proliferate to DRB1*0401 HV3 peptide. Conversely, people who express DRB1*1501 respond to DRB1*0401 HV3 peptide. Finally, people who express both DRB1*0401 (nonresponder haplotype) and DRB1*1501 (high responder haplotype) do not respond to DRB1 HV3 peptide, thus demonstrating tolerance. Therefore, the third hypervariable region of HLA-DRB1*0401 shapes the T cell repertoire.