CD8⁺ T-lymphocytes (T_CD8+) recognize minimal peptides of 8–10 residues which are the products of intracellularly processed proteins and are presented at the cell surface by major histocompatibility complex class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane, mediated by transporter associated with antigen-processing proteins or alternatively by endoplasmic reticulum-insertion signal sequences located at the NH₂-terminus of the precursor molecules. We report here that the addition of an endoplasmic reticulum-insertion signal sequence at the NH₂-terminus of T_CD8+ epitopes from chicken ovalbumin (amino acids 257–264) or a naturally occurring tumor antigen expressed by the murine mastocytoma P815 (P1A amino acids 35–43) significantly enhanced the priming of specific T_CD8+ in vivo. The signal sequence did not enhance peptide immunogenicity by merely increasing the hydrophobicity of the peptide, since ovalbumin amino acids 257–264 peptide with the signal sequence at its COOH-terminus did not demonstrate enhanced efficacy. The signal sequence did not act as a helper epitope, since T_CD8+ responses were not diminished in class II-deficient transgenic mice or in mice depleted of CD4⁺ T-cells in vivo. Importantly, a single immunization with the fusion peptide significantly prolonged survival of mice challenged with E.G7OVA, a thymoma transfected with the complementary DNA of chicken ovalbumin.