The prostatic epithelium is functionally organized in stem cell units. This unit consists of a slow turn over stem cell within the basal epithelial layer which can replenish itself and provide progeny which differentiate down either a neuroendocrine or exocrine pathway. The maturation along the exocrine pathway initially involves transit amplifying cells within the basal layer proliferating and subsequently the progeny maturing into intermediate cells. These intermediate cells migrate into the luminal layer where they terminally differentiate into non-proliferative secretory luminal cells which express prostate specific differentiation markers, like PSA. A growing body of experimental evidence has identified the proliferating transit amplifying/intermediate cells as the cells of origin for the common prostatic adenocarcinomas. Using a series of growth characteristics, and mRNA and protein markers, we have validated that primary cultures can be established in serum free defined media from surgically resected human prostates which are composed of essentially pure population of transit amplifying cells. At each serial passage, the subsequent cultures undergo enhanced maturation into intermediate cells and by the 7–10th passage these cells eventually lose their proliferative ability. This study validates that these cells are a useful and relevant system for the determination of molecular events involved in prostatic carcinogenesis. © 2003 Wiley-Liss, Inc.