The human telomerase reverse transcriptase hTERT is overexpressed in most human tumors and contributes importantly to oncogenesis by maintaining the integrity of telomeric DNA. Despite being a self-antigen, the hTERT enzyme is immunogenic. Peptides derived from hTERT have been shown both in vitro and in vivo to drive the activation and proliferation of peptide-specific T lymphocytes. An HLA-A2-binding peptide from hTERT (I540, ILAKFLHWL) has been used to generate peptide-specific T cells in vitro and in vivo in patients that lyse telomerase-positive tumors in an MHC-restricted fashion. Although these and other data suggest that I540 is naturally processed and presented on the surface of certain tumor cells, there are reports that I540-specific T cells, and in particular, T cell clones, do not lyse tumors in vitro. Here, we compared cytotoxic function of I540-specfic T cell clones vs. polyclonal T cell lines, including clones and lines generated from the same donor. We found that I540-specific polyclonal T cell lines lyse telomerase-positive tumors whereas non-specific polyclonal T cell lines and I540-specific T cell clones do not. Estimated TCR avidity for I540, as well as cell surface expression of CD45RO, CD45RA, CD28, CD27, CD57 and CD62L were similar between lines and clones. V beta usage, however, differed such that the majority of the I540-specific TCR repertoire found in polyclonal T cell lines was not represented in clones generated from the same source material. Thus, I540-specific T cells can vary in cytotoxic potential depending on the method of generation, isolation and expansion.