Prostate cancer and benign tumors of the prostate are the two most common neoplastic diseases in men in the United States, however, research on their causes and treatment has been slow because of the difficulty in obtaining fresh samples of human tissue and a lack of well characterized cell lines which exhibit growth and differentiation characteristics of normal prostatic epithelium. Non-neoplastic adult human prostatic epithelial cells from a white male donor were immortalized with human papillomavirus 18 which resulted in the establishment of the RWPE-1 cell line. Cells from the RWPE-1 cell line were further transformed by v-Ki-ras to establish the RWPE-2 cell line. The objectives of this study were to: (1) establish the prostatic epithelial origin and androgen responsiveness of RWPE-1 and RWPE-2 cell lines; (2) examine their response to growth factors; and (3) establish the malignant characteristics of the RWPE-2 cell line. Immunoperoxidase staining showed that both RWPE-1 and RWPE-2 cells express cytokeratins 8 and 18, which are characteristic of luminal prostatic epithelial cells, but they also coexpress basal cell cytokeratins. These cell lines show growth stimulation and prostate specific antigen (PSA) and androgen receptor (AR) expression in response to the synthetic androgen mibolerone, which establishes their prostatic epithelial origin. Both cell lines also show a dose-dependent growth stimulation by EGF and bFGF and growth inhibition when exposed to TGF-beta, however, the transformed RWPE-2 cells are less responsive. RWPE-1 cells neither grow in agar nor form tumors when injected into nude mice with or without Matrigel. However, RWPE-2 cells form colonies in agar and tumors in nude mice. In the in vitro invasion assay, RWPE-1 cells are not invasive whereas RWPE-2 cells are invasive. Nuclear expression of p53 and Rb proteins was heterogeneous but detectable by immunostaining in both cell lines. The RWPE-1 cells, which show many normal cell characteristics, and the malignant RWPE-2 cells, provide useful cell culture models for studies on prostate growth regulation and carcinogenesis.