Cytolytic CD8(+) T cells (CTL) are key to the immune response that controls virus infections and mediates disease protection. The ability of CTL to induce apoptosis of infected cells and/or limit viral replication is determined by recognition of processed viral peptide epitopes on the surface of the target cell. An understudied source of MHC class I-presented peptides is the aptly named "cryptic epitopes," defined by their nontraditional methods of generation, including derivation from alternative reading frames (ARFs). Although ARF-encoded epitopes have now been documented in a few systems, their potential functional relevance in vivo has been debated. In this study, we demonstrate the physiological significance of an ARF-derived CTL epitope in a retrovirus-induced disease model. We show that disease-susceptible CD8-deficient mice reconstituted with CTL specific for the retroviral ARF-derived SYNTGRFPPL epitope controlled an infection by the LP-BM5 retrovirus isolate, evidently at the level of viral clearance, resulting in protection of these mice from disease. These data indicate that ARF-derived epitopes are indeed relevant inducers of the immune system and demonstrate the importance of atypically generated peptides as functional Ag with a physiologic role in disease protection.