CTLs recognize peptide epitopes which are proteolytically generated by the proteasome and presented on MHC class I molecules. According to the defective ribosomal product (DRiP) hypothesis, epitopes originate from newly synthesized polypeptides which are degraded shortly after their translation. The DRiP hypothesis would explain how epitopes can be generated from long-lived proteins. We examined whether neosynthesis is required for presentation of the immunodominant epitope NP118 of the lymphocytic choriomeningitis virus nucleoprotein, which has a half-life of >3 days. Two days after nucleoprotein biosynthesis was terminated in a tetracycline-regulated transfectant, the presentation of the NP118 epitope ceased. This indicates that NP118 epitopes are generated from newly synthesized nucleoproteins rather than from the long-lived pool of nucleoproteins in the cell. Therefore, the lymphocytic choriomeningitis virus nucleoprotein is the first substrate for which a major prediction of the DRiP hypothesis, namely the requirement for neosynthesis, is shown to hold true.