The catalytic subunit of human telomerase reverse transcriptase (hTERT) is expressed in the majority of tumor cells of different histological origins as opposed to most normal somatic cells. This implicates hTERT as a widely expressed tumor-associated antigen and an attractive candidate for antigen-specific tumor immunotherapy. T lymphocytes specific for hTERT-derived epitopes have been isolated and shown reactive with hTERT-expressing tumor cells. To further increase the applicability of hTERT as a target antigen for immunotherapy, we set out to identify potential hTERT-derived, HLA-A1-restricted cytotoxic T-lymphocyte (CTL) epitopes. The "reverse immunology" approach, involving computer-assisted epitope prediction, in vitro CTL induction, and tetramer-guided CTL isolation, resulted in specific CTLs against hTERT-derived, HLA-A1-binding peptides. Intermediate- to low-avidity CTLs were induced against the hTERT325-333 peptide and recognized endogenously processed hTERT. Recognition of endogenous hTERT depended on an increase of hTERT expression above normal levels in tumor cells through hTERT transduction, most probably as a result of limited CTL avidity. The altered peptide ligand hTERT699T-707 was designed to increase HLA-A1-binding affinity of the hTERT699-707 peptide and was used to induce CTLs. However, these CTLs poorly cross-recognized native hTERT699-707 and failed to recognize endogenously processed hTERT. In conclusion, our study has identified the hTERT325-333 peptide as a potential hTERT-derived epitope that may prove useful for induction and monitoring of hTERT-specific, HLA-A1-restricted CTL responses.