Prostate cancer is the most common malignancy in men, and patients with metastatic disease have poor outcome even with the most advanced therapeutic approaches. Most cancer therapies target the bulk tumor cells, but may leave intact a small population of tumor-initiating cells (TICs), which are believed to be responsible for the subsequent relapse and metastasis. Using specific surface markers (CD44, integrin alpha(2)beta(1) and CD133), Hoechst 33342 dye exclusion, and holoclone formation, we isolated TICs from a panel of prostate cancer cell lines (DU145, C4-2 and LNCaP). We have found that prostate TICs have significant telomerase activity which is inhibited by imetelstat sodium (GRN163L), a new telomerase antagonist that is currently in Phase I/II clinical trials for several hematological and solid tumor malignancies. Prostate TICs telomeres were of similar average length to the telomeres of the main population of cells and significant telomere shortening was detected in prostate TICs as a result of imetelstat treatment. These findings suggest that telomerase inhibition therapy may be able to efficiently target the prostate TICs in addition to the bulk tumor cells, providing new opportunities for combination therapies.