Background: Protein antigens are presented to cytotoxic T lymphocytes as small peptides (approximately 9–10 amino acids long) bound to class I molecules of the major histocompatibility complex. The identification of tumor-associated antigens and specific peptide epitopes (i.e., antigenic determinants) may be useful in the development of anticancer vaccines. The generation of a cytotoxic T-cell response to one peptide epitope (amino acids 146–154) of human prostatespecific antigen (PSA) has been reported. Purpose: Our aim was to identify novel PSA peptides capable of eliciting specific cytotoxic T-cell responses. Methods: Candidate peptides were identified on the basis of the following criteria: 1) they contained consensus amino acid motifs for binding to HLAA2, which is the most common type of class I molecule; 2) they lacked strong homology with PSA-related kallikrein proteins; and 3) they were capable of stabilizing HLA-A2 class I molecules on the surface of human T2 (transport deletion mutant) cells, which are defective in antigen presentation. T-cell lines capable of killing (i.e., lysing) T2 target cells that had been pulsed with specific PSA peptides were generated from three different males (two disease-free individuals and one patient with prostate cancer) by incubating peripheral blood mononuclear cells with the peptides and interleukin 2. Specific cell lysis was monitored by the release of radioactivity from target cells that had been labeled with [ ¹¹¹ In]oxyquinoline. Results: Two novel PSA peptides capable of eliciting cytotoxic T-cell responses were identified; these peptides were designated PSA-1 (amino acids 141–150) and PSA-3 (amino acids 154–163). Four different cytotoxic T-cell lines were generated in response to these peptides—three against PSA-3 and one against PSA-1. Specific lysis of peptide-pulsed T2 cells by the T-cell lines was blocked by the addition of a monoclonal antibody directed against class I molecules. The T-cell lines were also capable of lysing PSA-positive, HLA-A2-positive LNCaP cells (human prostate carcinoma cells). The specificity of LNCaP cell lysis was shown by the following: 1) the inability of added human K562 (chronic myelogenous leukemia) cells to inhibit it, 2) the ability of added anti-HLA-A2 antibodies to block it, and 3) the inability of the T-cell lines to induce substantial lysis of PSA-negative, HLA-A2-positive human cancer cells. Implications: Our studies form a rational basis for the use of PSA peptides or recombinant vectors encoding PSA in the development of anticancer vaccine immunotherapy protocols for patients with prostate cancer.