The expression of specific alleles of the human HLA-DR locus is associated with increased risk for the development of rheumatoid arthritis. Examination of the amino acid sequence of the DR beta chain has revealed that risk for RA correlates with a cluster of polymorphic residues located between positions 67 and 86, and in particular with the identity of residues 70, 71, and 86. To examine the contributions of these HLA-DR polymorphic residues to antigen-specific T cell responses, the DRB1*0401 gene was subjected to site-directed mutagenesis and forms possessing alanine in place of the naturally occurring amino acid at positions 70, 71, 86, and 70/71 were generated. The mutated genes were coexpressed with the DRA gene in Chinese hamster ovary cells and the transfectants were tested as stimulator cells for a panel of three human influenza virus hemagglutinin-specific T cell clones. Additionally, soluble forms of the mutant DR molecules were examined for their ability to bind peptide. All of the mutants had a modest loss of affinity for the peptide relative to the wild type, but there were no significant differences in peptide binding ability among the substituted molecules. In contrast to the relatively uniform influence on peptide binding, the impact of these mutations on T cell stimulation was heterogeneous. Specifically, these studies indicate that residue 71 plays a critical role in T cell stimulation either through direct contact with the T cell receptor or by changing the orientation or conformation of the peptide-MHC complex. Replacement of residue 71 with alanine abrogated stimulation of all of the T cell clones. Two of three clones were affected by changes at residue 70 while none lost recognition when amino acid 86 was converted from Val to Ala. These data emphasize that subtle alterations in structure can have a profound impact on T cell recognition.