A synthetic peptide E474 SFAVATTAL, derived from the sequence of mouse mammary tumor virus envelope protein, was previously shown to bind class I MHC Kd. Immunization of BALB/c mice with E474 in 50% incomplete Freund's adjuvant (IFA) followed by in vitro stimulation of immune cells with E474-coated antigen-presenting cells resulted in peptide-specific cytotoxic T lymphocytes (CTL). Furthermore, anti-E474 CTL lysed mammary tumor cell lines D2F2 and D2A1, derived from a spontaneous tumor that arose in BALB/c pre-neoplastic hyperplastic alveolar nodule (HAN) D2 line. Expression of Kd by D2A1 and D2F2 cells was verified by flow cytometry, and lysis of D2 tumor cells was blocked by monoclonal antibody 31-34-S, which interacted with the peptide-binding region of Kd, supporting the recognition of E474 in Kd by anti-E474 CTL. Immunization of BALB/c mice with E474 before D2F2 tumor challenge resulted in reduced tumor growth. Therefore, E474 is naturally processed and presented by these tumor cells and can induce anti-tumor immunity.