Understanding normal and cancer stem cells may provide insight into the origin of and new therapeutics for prostate cancer. Normal and cancer stem cells in prostate have recently been identified with a CD44⁺/α₂β₁^high/CD133⁺ phenotype. Stromal cell–derived factor-1 (SDF-1) and its receptor, CXCR4, have multiple essential functions, including homing of stem cells and metastasis of cancer cells. We show here that human telomerase reverse transcriptase (hTERT)-immortalized primary nonmalignant (RC-165N/hTERT) and malignant (RC-92a/hTERT) tumor-derived human prostate epithelial cell lines retain stem cell properties with a CD133⁺/CD44⁺/α₂β₁⁺/34βE12⁺/CK18⁺/p63⁻/androgen receptor (AR)⁻/PSA⁻ phenotype. Higher CD133 expression was detected in the hTERT-immortalized cells than in primary prostate cells. These immortalized cells exhibited “prostaspheres” in nonadherent culture systems and also maintained higher CD133 expression. The CD133⁺ cells from these immortalized cell lines had high proliferative potential and were able to differentiate into AR⁺ phenotype. In three-dimensional culture, the CD133⁺ cells from RC-165N/hTERT cells produced branched structures, whereas the CD133⁺ cells from RC-92a/hTERT cells produced large irregular spheroids with less branched structures. SDF-1 induced, but anti-CXCR4 antibody inhibited, migration of CD133⁺ cells from RC-92a/hTERT cells, which coexpressed CXCR4. CXCR4/SDF-1 may sustain tumor chemotaxis in cancer stem cells. Furthermore, immunostaining of clinical prostate specimens showed that CD133 expression was detected in a subpopulation of prostate cancer cells and corresponded to the loss of AR. Expression of CXCR4 was also detected in CD133⁺ cancer cells. These novel in vitro models may offer useful tools for the study of the biological features and functional integration of normal and cancer stem cells in prostate. [Cancer Res 2007;67(7):3153–61]