The association between HLA-DR4 and rheumatoid arthritis (RA) has been established in many ethnic groups. To clarify the determinant of susceptibility to RA, a polymorphic segment of the HLA-DRB gene was amplified in vitro by polymerase chain reaction and analyzed with oligonucleotide probes specific for the HLA-DR4 DNA sequences. A particular sequence encoding amino acids Gln70-Arg71-Arg72-Ala73-Ala74 showed a strong association with RA (p less than 0.005, relative risk 6.0). This amino acid sequence occurs in the DRB molecules with three RA-associated specificities, DR4/Dw14, DR4/Dw15, and DR1. DR4/Dw4, which is common in Caucasian RA patients, has a strikingly similar amino acid sequence Gln70-Lys71-Arg72-Ala73-Ala74 in terms of polarity and charge profiles. Other RA nonassociated sequences differ from this sequence by at least one amino acid substitution that causes the change of the net charge. The composition of amino acid residues at the positions 70-74 may play a crucial role in the pathogenesis of RA.