The aim of this study was to investigate the best administration of telomerase reverse transcriptase (TERT572), an human leukocyte antigen-A*0201-restricted cryptic epitope of telomerase, and its optimized variant TERT(572Y) to elicit specific T cell immune responses in cancer patients. Forty-eight cancer patients with chemo-resistant tumors received 2 subcutaneous injections of TERT(572Y) at 2 mg followed at random by 4 subcutaneous injections of either TERT572 or TERT(572Y) peptides at 2 mg every 3 weeks. Specific immune response was evaluated by interferon-γ enzyme-linked immunosorbent spot. T cell responses after the sixth vaccination were detected more frequently (44% vs. 17%), and with higher number of peptide-specific reactive T cells (60 T cells/2 × 10(5) peripheral blood mononuclear cell vs. 10 T cells/2 × 10(5) peripheral blood mononuclear cell, P=0.04), and higher avidity in the patients who received 4 more vaccinations with the TERT572 peptide compared with patients who received only TERT(572Y) vaccinations. These results demonstrate that the best vaccination schedule involves first the administration of the optimized TERT(572Y) followed by the native TERT572 peptides in patients who are candidates for cancer immunotherapy.