Human histocompatibility leukocyte antigen E (HLA‐E) and mouse major histocompatibility complex (MHC) class Ib antigen, Qa‐1, share the same substitutions at two normally conserved positions 143 and 147, which are likely to affect binding of the C terminus of peptides. Qa‐1 is able to bind a peptide derived from the leader sequence of H‐2 D and H‐2 L molecules. We developed a peptide binding assay in vitro to compare the binding specificity of HLA‐E with the mouse MHC class Ib molecule Qa‐1. We demonstrate that HLA‐E binds, although poorly, the peptide which binds to Qa‐1 and that it also binds nonamer signal sequence‐derived peptides from human MHC class I molecules. Using alanine and glycine substitutions, we could define primary anchor residues at positions 2 and 9 and secondary anchor residues at position 7 and possibly 3.