Summary Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides in association with class I MHC molecules at the cell surface. Newly synthesized viral polypeptides are processed in the cytoplasm and the fragments of antigen are transported into the endoplasmic reticulum (ER) via a peptide transporter where they complex with nascent class I molecules. The peptide‐MHC complex is transported to the cell surface and presented to CTL. Sequence analysis of endogenously expressed, MHC‐associated self or viral antigens indicates that the naturally processed peptides bound to class I MHC molecules are in general 9 ± 1 residues long. Peptides bound to specific class I MHC molecules have in common allele‐specific motifs of conserved residues. The motif for the class I K d molecules has been shown to be nine or 10 residues with the sequence X‐Tyr‐(X)6‐I/L or X‐Tyr‐(X)7‐I/L. The Tyr residue at the second position and the I/L residue at the ninth position are allele‐specific anchor residues which appear to be required for binding of the peptide to K d . To examine the stringency of the requirement for Tyr at the second position, we have performed saturation mutagenesis of a minigene encoding the class I K d ‐restricted influenza HA210‐219 site at the Tyr residue 211. A series of 10 mutants was tested for effects on target‐cell sensitization. Most amino acid substitutions for the Tyr residue resulted in a loss of endogenous peptide recognition by HA210‐219 reactive CTL, consistent with the critical role of the Tyr at the second position for interaction with K d molecules. One mutant gene‐product encoding a His substitution for the Tyr residue was recognized by CTL. However, the corresponding synthetic peptide containing a His substitution at the dominant anchor position bound only weakly to K d , and target cells treated with the peptide were poorly recognized by CTL. The endogenous His‐containing peptide was also less stably associated with class I MHC K d molecules at the cell surface than the wild‐type Tyr peptide. These data indicate that endogenous antigenic peptides may bind newly‐synthesized class I MHC molecules in the ER more efficiently than fully formed class I molecules at the cell surface and that endogenous peptides may dissociate from class I MHC molecules at different rates. The implications of these findings for CTL recognition and epitope mapping are discussed.