Recognition of virus-infected or transformed cells by CD8+ CTL requires a trimolecular complex composed of MHC class I, beta2-microglobulin, and a specific foreign peptide composed of 8 to 10 linear amino acids. The generation of such CTL epitopes has traditionally been thought to be from the primary open reading frame encoding the viral or tumor-associated proteins. In this report it is demonstrated that a viral CTL epitope can also be generated from an alternative reading frame. Using a combination of synthetic peptides and Sindbis or vaccinia expression systems, MHC class I Kd-restricted BALB/cByJ CTL directed against defective gag gene constructs of the LP-BM5 virus complex that causes murine AIDS were shown to have specificity for the antigenic peptide SYNTGRFPPL. This epitope is generated in a novel fashion from the second open reading frame (ORF2) of both the defective and ecotropic helper virus components of LP-BM5. Importantly, lysis of target cells expressing BM5 ecotropic helper, and/or defective viral gag, demonstrated that the SYNTGRFPPL epitope is generated during the course of a normal retroviral infection. Furthermore, MAIDS-resistant BALB/cByJ mice also generated secondary restimulated CTL specific for SYNTGRFPPL following in vivo priming with the LP-BM5 retroviral complex. These data suggest that retroviruses, and potentially other viruses and foreign genes, are capable of expressing T cell epitopes from alternative open reading frames. If one considers the influence of self peptides on T cell development, these "alternative reading frame-derived" peptides could provide an important additional influence on the functional T cell repertoire.