The first hydrophobic pocket, P1, of class II MHC has been shown to be an important site of peptide anchoring. Two polymorphisms occur in this pocket in the human class II MHC beta chain at position 85 and 86. beta 85 is usually Val, occasionally Ala, whereas beta 86 can be Gly or Val. However, Ala85 is found only in conjunction with Val86. The independent effect of the polymorphism at these two positions on the binding of normal and substituted antigenic peptides has never been examined. To do so, three soluble HLA-DR1 variants that contain the naturally occurring combinations of these side chains at these two positions were generated and tested with a panel of influenza matrix peptides varying at anchor P1. DR1 alleles differing only at position 86 are very similar in the binding of a panel of antigenic peptide, indicating that beta 86 does not substantially influence the peptide binding of DR1. In contrast, DR1 varying only at position beta 85 differ in their binding of substituted peptides containing Ala, Tyr or Trp at the P1 anchor position. Thus, beta 85 shows the predominant effect on the P1 anchor side chain preference of the P1 pocket in DR1. This is in contrast to other HLA-DR alleles where beta 86 has been shown to control the nature of the P1 anchor. These previous data together with our own imply that the role of polymorphism in P1 may be influenced by the contextual framework of the remaining allelic polymorphism.