We have studied polypeptide processing by purified proteasomes, with regard to proteolytic specificity and cytotoxic T-lymphocyte (CTL) epitope generation. Owing to defined preferences with respect to cleavage sites and fragment length, proteasomes degrade polypeptide substrates into cohorts of overlapping oligopeptides. Many of the proteolytic fragments exhibit structural features in common with major histocompatibility complex (MHC) class I ligands including fragment size and frequencies of amino acids at fragment boundaries. Proteasomes frequently generate definitive MHC class I ligands and/or slightly longer peptides, while substantially larger peptides are rare. Individual CTL epitopes are produced in widely varying amounts, often consistent with immunohierarchies among CTL epitopes. We further found that polypeptide processing is remarkably conserved among proteasomes of eukaryotic origin and that invertebrate proteasomes can efficiently produce known high-copy MHC class I ligands, suggesting evolutionary adaptation of the transporter associated with antigen processing and MHC class I to ancient constraints imposed by proteasomal protein degradation.