Motifs of peptides naturally associated with H-2Ek and Ed molecules were determined by (I) pool sequencing of natural ligand mixtures and (II) sequencing of individual natural ligands followed by their alignment to the basic motif suggested by pool sequencing. The data reveal nine amino acid motifs with interaction sites at relative positions P1, P4, P6 and P9, with specificities that are identical at some but different at other anchor positions between Ed and Ek motifs, illustrating the different requirements for peptides to be presented by these two MHC molecules. The anchors with the most restricted specificity are P1 and P9. P1 is aliphatic for Ek and predominantly aromatic for Ed. P9 is positively charged for both molecules. P4 and P6 show a totally different amino acid preference between Ek and Ed ligand motifs. An alignment of Ed and Ek protein sequences to the recently reported HLA-DR1 pocket residues is in agreement with observed anchor residues in Ek and Ed motifs, thus confirming the predicted similarity of mouse class II E molecules with human DR molecules. Furthermore, this alignment was extended to the putative pockets of class II Eb and Ek molecules, and allowed, together with sequence information of previously identified natural ligands of Eb and Ec molecules, a prediction of their respective motifs. The information obtained by this study should be useful to identify putative class II E epitopes in proteins and to design peptides for blocking class II E molecules.