The previously defined binding motif of MHC class I H-2Kd-restricted antigenic peptides consists of a Y residue in position P2 and a hydrophobic residue with a large aliphatic side chain (L, I, or V) in position P9/P10 of optimal 9- or 10-mer peptides. We show now that the presence of a charged or a F residue in position P5 reduces the Kd-restricted competitor activity of several cytotoxic T lymphocyte (CTL) epitopes and model peptides, at a degree comparable to A substitutions for the P2 or the P9/P10 anchor residues. Various charged, polar, aromatic, and aliphatic amino acids were substituted for S256 in the CTL epitope Plasmodium berghei circumsporozoite (CS) 253-260 8-mer and in its CS 252-260 9-mer form, whereas a more restricted panel of substitutions was tested in the CTL epitopes influenza nucleoprotein 147-155 9-mer and HLA-CW3 170-179 10-mer. Analysis of all the Kd-restricted epitopes so far defined also revealed an uncharged residue at this position. These additional structural constraints present in the Kd binding motif may thus improve the prediction of new epitopes recognized by T cells in the context of this MHC molecule.