Sequence-independent interactions involving the free peptidic NH 2 terminus are thought to be an essential feature of peptide binding to classical major histocompatibility complex (MHC) class I proteins. Challenging this paradigm, a natural Nα -acetylated ligand of human histocompatibility leukocyte antigen (HLA)-B39 was identified in this study. It matched the NH 2 -terminal sequence of two human helicases, was resistant to aminopeptidase M, and was produced with high yield from a synthetic 30 mer with the sequence of the putative parental protein by the 20S proteasome. This is the first reported natural ligand of classical MHC class I antigens that has a blocked NH 2 terminus.