The generation of suitable in vitro models is critical for understanding the process associated with the development and progression of prostate cancer in high-risk African-American men. However, the generation of long-term human prostate epithelial cell lines derived from primary human prostate epithelium have been unsuccessful due to the absence of in vitro immortalization. We have successfully established an immortal human prostate epithelial cell line from primary benign tissues of African-American prostate cancer patients by using telomerase. The actively proliferating secondary African-American prostate epithelial RC-165N cells, derived from benign prostate tissue of a radical prostatectomy specimen, were transduced through infection with a retrovirus vector expressing the human telomerase catalytic subunit (hTERT). A high level of telomerase activity was detected in RC-165N/hTERT cells but not in RC-165N cells. RC-165N/hTERT cells are currently growing well at passage 50 whereas RC-165N cells senesced within passage 3. RC-165N/hTERT cells exhibit epithelial morphology. These immortalized cells showed no cell growth in soft agar, and no tumor formation in SCID mice. The RC-165N/hTERT cells express androgen-regulated prostate-specific homobox gene. NKX 3.1 and epithelial cell specific cytokeratin 8, androgen receptor (AR), prostate stem cell antigen and p16, but not PSA. AR protein was detected by Western blot analysis.