Prostate cancer is the most common male cancer in the United States. Research on the mechanisms of prostate cancer progression has been limited by the lack of suitable in vitro systems. A hurdle in understanding the molecular genetic changes in prostate cancer has been the difficulty in establishing premalignant lesions and primary prostate tumors as in vitro cell cultures. Primary prostate epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we and others have developed in vitro models of prostate epithelial cell immortalization. Methods are described for the processing of primary human prostate biopsy samples and the generation of human prostate epithelial (HPE) cells in serum-free conditions. Retrovirus containing human telomerase reverse transcriptase (hTERT) is used for the immortalization of primary HPE cells, and the methods for the characterization of HPE cell lines are discussed. These in vitro prostate cell culture models are useful for the study of prostate normal and cancer stem cells, are critical for defining the mechanisms of prostate cancer progression and for testing preventive and therapeutic regimens.