CD8+ cytotoxic T cells recognize their targets by the presence of unique peptide bound to a major histocompatibility complex (MHC) class I molecules on the cell surface. The MHC molecules normally display thousands of distinct peptides, making it difficult to identify individual antigenic peptides, their protein precursors, and their relative importance in the T-cell response. Here we used the EL-4 tumor-specific lacZ-inducible KZ30.6 T cell as a probe for detecting the peptide/MHC ligand that was generated in cells transfected with an EL-4 cDNA library. These expression screens allowed identification of a mouse mammary tumor virus (MMTV) transcript as the source of the antigenic peptide presented by the Kb MHC molecule. The antigenic activity was encoded within the MMTV env gene and was defined by the octapeptide ANYDFICV (AFV8). Synthetic AFV8 stimulated KZ30.6 T cells at picomolar concentrations and coeluted with one of two active peptides in HPLC-fractionated extracts of EL-4 cells. The AFV8/Kb complex was also recognized by two other EL-4-specific T cells. The results illustrate a novel strategy for identifying T-cell-stimulating antigens and suggest that the MMTV env gene and its naturally processed AFV8 peptide product can serve as a model for study of antigen processing and tumor immunotherapy.