Peptide epitopes presented through class I major histocompatability complex (MHC class I) on the cell surface, are generated by proteolytic processing of proteinantigens in the cytoplasm. The length and amino acid sequence determine whether a given peptide can fit into the peptide binding groove of class I heavy chain molecules and subsequently be presented to the immune system. The mode of action of the processing pathway is therefore of great interest. To study the processing mechanism of MHC class I-restricted intracellular antigens, we reconstituted the proteolytic processing of a model antigen in a cell-free system. Incubation of oxidized and urea-treated OVA in lymphocyte lysate resulted in partial degradation of the antigen. Degradation of the antigen depended on the presence of ATP. Addition of methylated ubiquitin abolished the reaction which was then restored by addition of an excess of native ubiquitin, indicating that the breakdown of the antigen in lymphocyte lysate is mediated by the ubiquitin proteolytic system. Upon incubation of modified OVA in lymphocyte lysate, a specific antigenic peptide was generated. The peptide was recognized by cytotoxic T lymphocytes directed against OVA-derived, H-2K b -restricted peptide (SIINFEKL), and by a monoclonal antibody that recognizes cell-bound K b -SIINFEKL complexes. Formation of the peptide epitope depended on the presence of ATP and ubiquitin. These results indicate that proteolytic processing of modified OVA is carried out by the ubiquitin-mediated degradation system. The experimental system described provides a tool to analyze the molecular mechanisms underlying the generation of specific, MHC class I-restricted peptide epitopes.Cells display foreign and altered intracellular protein-antigens to cytotoxic T-lymphocytes through MHC 1 class I molecules. Intracellular antigens are not presented directly. The intracellular antigen is first proteolyzed in the cytoplasm of the antigen presenting cell to yield short peptides. Suitable peptides are then translocated through specialized peptide transporters (termed TAP) into the lumen of the endoplasmic reticulum where they bind to newly synthesized MHC class I molecules. The entire complex is then stabilized and transported to the plasma membrane where it is presented to the immune system (1).Proteolysis and generation of antigenic peptides is carried out by the multicatalytic protease (26 S proteasome). Direct evidence for the involvement of the proteasome in the processing of MHC class-I restricted intracellular antigens came from experiments in which membrane-permeable inhibitors of proteasomes were added to cells and the capacity of these cells to present antigens was monitored. These inhibitors, which block proteasome activity in vitro, inhibited the cellular turnover of short-lived and long-lived proteins, assembly of class I molecules as well as presentation of OVA introduced into the cytoplasm (2).The proteasome constitutes the major proteolytic activity in the cytosol and nucleus of all eukaryotes (3)...