BACKGROUND. Human prostate specific antigen (PSA) is expressed selectively in prostate epithelium and is a potential target for the immunotherapy against prostate cancer. Various PSA-based vaccines have been reported to induce cytotoxic T lymphocyte (CTL) responses in animal models. Here, we present the identification and validation of an immunodominant CTL epitope of PSA in C57B1/6 mice (H-2 ^b). METHODS. PSA-specific CTLs were induced by immunization with a plasmid expressing PSA. Epitope specificity of the CTLs was determined by their reactivity against a panel of C-terminus truncated or mutated PSA proteins and use of bioinformatical prediction with the SYFPEITHI algorithm. RESULTS. The majority of PSA-specific CTLs were directed against a single H-2D ^b restricted epitope corresponding to the amino acid residues 65-74 (HCIRNKSVIL) of the protein. The CTLs had similar functional avidity against two putative H-2D ^b binding peptides: a 9-aa-long psa65-73 (HCIRNKSVI) and a 10-aa-long psa65-74 (HCIRNKSVIL). CONCLUSIONS. We demonstrate that the psa65-73 peptide can be used for reactivation of PSA-specific CTLs in vitro and ex vivo, and H-2D ^b pentamers assembled with this peptide are an efficient tool for monitoring of PSA-specific CTL responses after DNA vaccination.