CD8+ T lymphocytes (TCD8+) play an important role in cellular immune responses. TCD8+ recognize MHC class I molecules complexed to peptides of 8 to 10 residues derived largely from cytosolic proteins. Proteins are generally thought to be fragmented in the cytoplasm and delivered to nascent class I molecules in the endoplasmic reticulum (ER) by a peptide transporter encoded by the MHC. To explore the extent to which TCD8+ induction in vivo is limited by proteolysis or peptide transport into the ER, mice were immunized with recombinant vaccinia viruses containing mini-genes encoding antigenic peptides (bypassing the need for proteolysis), or these peptides with a NH2-terminal ER insertion sequence (bypassing the requirements for both proteolysis and transport). Additionally, mice were immunized with recombinant vaccinia viruses encoding rapidly degraded fragments of proteins. We report that limitations in induction of TCD8+ responses vary among Ags: for some, full length proteins are as immunogenic as other forms tested; for others, maximal responses are induced by peptides or by peptides targeted to the ER. Most importantly, in every circumstance examined, targeting peptides to the ER never diminished, and in some cases greatly enhanced, the TCD8+ immune response and provide an important alternative strategy in the design of live viral or naked DNA vaccines for the treatment of cancer and infectious diseases.