Cytotoxic T lymphocytes (CTLs) recognize peptide antigens associated with cell surface major histocompatibility complex (MHC) molecules. The identification of tumor cell-derived peptides capable of eliciting anti-tumor CTL responses would enable the design of antigen-specific immunotherapies. Our strategy to identify such potentially therapeutic peptides relies on selecting high-affinity MHC binders from known tumor-associated antigens. These peptides are subsequently tested for their ability to induce CTLs capable of killing tumor cells. With this strategy, we have identified a nine-residue epitope, derived from the product of the tumor-associated gene MAGE-3, which has the capacity to induce in vitro CTLs that kill melanoma and other tumor cell lines. These results show the primary in vitro induction of tumor-specific human CTLs and illustrate the feasibility of ex vivo antigen-specific approaches to the immunological therapy of cancer.