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Although estrogens have been long implicated in the prostate carcinogenesis, direct evidence showing their carcinogenicity on prostatic epithelial cells has not yet been clearly demonstrated. In this study, we treated an immortalized, non-transformed and androgen-responsive rat prostatic epithelial cell line NRP-152 with 17β-estradiol (E₂) at concentrations 1–3μM for period 2–6weeks. After in vitro treatment, we evaluated the anchorage-independent growth of E₂-treated NRP-152 cells by soft agar assay and isolated the colonies formed by the transformed E₂-NRP-152 cells in soft agar for further growth phenotype characterization. Our results showed that the isolated E₂-NRP-152 clones displayed neoplastic transformation phenotype, as demonstrated by their capacity of forming colonies in soft agar and tumors in immunodeficient nude mice, while losing their spheroid formation capacity in Matrigel 3D-culture …