It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues. However, due to lack of specific stem cell surface markers, CSCs are very difficult to be separated from some cancer cells, which becomes the key barrier of functional studies of CSCs. The sorting method by side population cells (SP) lays a solid foundation for in-depth and comprehensive study of CSCs. To identify the existence of SP in prostate cancer cell lines, we applied flow cytometry sorting by SP to cultures of prostate cancer cell lines (TSU, LnCap, and PC-3), and the cancer stem-like characteristics of SP were verified through experiments in vitro and in vivo. The proportion of SP in TSU cells was calculated to be 1.60%+/- 0.40% , and that in PC-3 and LnCap cells was calculated to be 0.80%+/- 0.05% and 0.60%+/- 0.20%, respectively. The colony formation assay demonstrated that the colony formation rate of SP to non-SP sorted from TSU via flow cytometry was 0.495 +/- 0.038 to 0.177 +/- 0.029 in 500 cells, 0.505 +/- 0.026 to 0.169 +/- 0.024 in 250 cells, and 0.088 +/- 0.016 to 0.043 +/- 0.012 in 125 cells respectively. In the in vivo experiments, tumors were observed in all the mice on the 10th day after injecting 50 000 cells subcutaneously in SP group, whereas when 5x10(6) cells were injected in non-SP group, tumors were developed in only 4 out of 8 mice until the 3rd week before the end of the experiment. Our results revealed that prostate cancer cells contain a small subset of cells, called SP, possessing much greater capacity of colony formation and tumorigenic potential than non-SP. These suggest that SP in prostate cancer cells may play a key role in the self-renewal and proliferation, and have the characteristics of cancer stem-like cells. Dissecting these features will provide a new understanding of the function of prostate CSCs in tumorigenicity and transformation.