The recent cloning of tumor-associated antigens (TAAs) recognized by CD8+ T lymphocytes (TCD8+) has made it possible to use recombinant and synthetic forms of TAAs to generate TCD8+ with anti-tumor activity. To explore new therapeutic strategies in a mouse model, we retrovirally transduced the experimental murine tumor CT26(H-2d), with the lacZ gene encoding our model TAA, beta-galactosidase (beta-gal). The transduced cell line, CT26.CL25, grew as rapidly and as lethally as the parental cell line in normal, immunocompetent animals. In an attempt to elicit TCD8+ directed against our model TAA by using purely recombinant and synthetic forms of our model TAA, we synthesized a nine-amino-acid long immunodominant peptide of beta-gal (TPH-PARIGL), corresponding to amino acid residues 876-884, which was known to be presented by the Ld major histocompatibility complex (MHC) class I molecule, and a recombinant vaccinia virus encoding the full-length beta-gal protein (VJS6). Splenocytes obtained from naïve mice and co-cultured with beta-gal peptide could not be expanded in primary ex vivo cultures. However, mice immunized with VJS6, but not with a control recombinant vaccinia virus, yielded splenocytes that were capable of specifically lysing CT26.CL25 in vitro after co-culture with beta-gal peptide. Most significantly, adoptive transfer of these cells could effectively treat mice bearing 3-day-old established pulmonary metastases. These observations show that therapeutic TCD8+ directed against a model TAA could be generated by using purely recombinant and synthetic forms of this antigen. These findings point the way to a potentially useful immunotherapeutic strategy, which has been made possible by the recent cloning of immunogenic TAAs that are expressed by human malignancies.