The proteasome generates exact major histocompatibility complex (MHC) class I ligands as well as NH 2-terminal-extended precursor peptides. The proteases responsible for the final NH 2-terminal trimming of the precursor peptides had, until now, not been determined. By using specific selective criteria we purified two cytosolic proteolytic activities, puromycin-sensitive aminopeptidase and bleomycin hydrolase. These proteases could remove NH 2-terminal amino acids from the vesicular stomatitis virus nucleoprotein cytotoxic T cell epitope 52–59 (RGYVYQGL) resulting, in combination with proteasomes, in the generation of the correct epitope. Our data provide evidence for the existence of redundant systems acting downstream of the proteasome in the antigen-processing pathway for MHC class I molecules.