The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild-type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA-A2.1 in an in vitro assay. These HLA-A2.1-binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing-defective cell line (174CEM.T2) as antigen-presenting cell. These cells display "empty" HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8+ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild-type or tumor-specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.