Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1–7 additional N-terminal residues. Only 6–8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-γ-induced β-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2–4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (eg the interferon-γ-induced enzyme leucine aminopeptidase).