Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.