Prostate cancer is a major health concern in the Western world. Prostate cancer stem cells have been implicated to be involved in, if not solely responsible for prostate cancer initiation and relapse after surgical, hormonal, and chemotherapy. Until now, the identity of the presumed prostate cancer stem cell has been illusive and the efforts to characterize such cells have been hampered by the lack of efficient prostate stem cell activity assay. Anchorage-independent growth of brain and mammary stem cells in vitro has been proved to be a useful tool to enrich and characterize neural and mammary stem cells. Recently, we have successfully established a prostosphere cultivation method employing strategies similar to neurosphere and mammosphere cultures. Our results demonstrate that prostosphere-forming cells possess stem cell characters and are significantly over-represented in cancer tissues.