Relative affinities were determined for the interaction of H-2Db with all the peptides from the A/PR/8/34 strain of influenza virus that contained the Db-binding motif. The results indicated that, even though 23 peptides with the appropriate motif were identified and analysed, binding of only five of them could be detected at peptide concentrations lower than 10(-7) M. Of these five, only one, TGICNQNII, bound with better affinity than the nucleoprotein-derived natural epitope, ASNENMETM. The origin of the higher binding peptide was the influenza neuraminidase, a protein for which little cytosolic processing would be expected since it is a surface glycoprotein. To establish why many of the influenza-derived peptides did not bind, the role of non-anchor residues on Db-peptide interactions was analysed, using a scheme where QDIENEEKI, a non-binding peptide from the influenza virus polymerase 1, was sequentially converted to ASNENMETI, which binds to Db with an affinity similar to that of ASNENMETM. Although all positions examined influenced peptide binding, peptide residue no. 2 (P2) was of particular importance. Therefore, each of the 20 naturally occurring amino acids were inserted at this position to investigate their effects on peptide-MHC interaction. The results indicated that amino acids having side chains with charged or ring structures were deleterious, while non-polar and polar residues were either neutral or facilitated binding to different degrees. Our data also indicated that every residue of the peptide contributes to the stability of the MHC-peptide complex, and the final affinity is dependent on the nature of the amino acids at each position, not just on those at a small number of anchor positions. The results also suggested that increased stability, as indicated by the half-life of the peptide-MHC class I complex, might play an important role in selecting the immunodominant epitope.