The valine-glycine dimorphism at position 86 of the DR beta-chain exhibited by most DR alleles has been shown to affect peptide binding. We demonstrate that DR1-restricted antigenic peptides differ in the extent to which binding is affected by amino acid substitution at positions 85 and 86 of the DR1 beta-chain. Binding of peptides derived from influenza hemagglutinin (HA306-320) and tetanus toxin (TT830-843) but not influenza matrix protein (MP19-31) was diminished on cells expressing DR1 beta-chains encoded by DRB1*0102 relative to DRB1*0101. The presence of tyrosine within HA306-320 and TT830-843 vs leucine within MP19-31 at a single DR contact position was revealed by alignment of the peptides according to a DR-binding motif. HA306-320 bearing leucine at this position (HA 306-320L309) bound to DR1 possessing either DRB1*0101- or DRB1*0102-encoded beta-chains suggesting that DR residues may discriminate among peptides based upon amino acid identity at a single position within the peptide. Furthermore, whereas all HA306-320-specific T cell clones recognized HA306-320L309 in the context of DR1 molecules possessing DRB1*0102-encoded beta-chains, some T cell clones failed to recognize HA306-320L309 in the context of DR1 molecules possessing DRB1*0101-encoded beta-chains. These results suggest that peptide conformation may also be affected by amino acid substitution at positions 85 and or 86 of the DR1 beta-chain.