Invasive prostatic carcinomas and prostatic intraepithelial neoplasia (PIN) are characterized by a loss of normal cell organization, cell polarity, and cell:cell and cell:basement membrane adhesion. The objective of this study was to establish in vitro three-dimensional (3-D) cell models which can be used to investigate mechanisms involved in acinar morphogenesis and differentiation in normal prostatic epithelium and their abnormalities in cancer cells. The process of acinar morphogenesis, including structural and functional differentiation, was investigated by culture on basement membrane gels (Matrigel). The human papillomavirus 18 immortalized, non-tumorigenic cell line RWPE-1, the v-Ki-ras transformed, tumorigenic RWPE-2 cell line derived from RWPE-1 cells (see previous paper pp. 1221-1229) and the human prostatic carcinoma cell line DU-145 were used. When cultured on Matrigel, RWPE-2 cells remain as single cells or form small aggregates and DU-145 cells form large amorphous cell aggregates without any organization or lumen. In contrast, RWPE-1 cells form acini of polarized epithelium with a distinct lumen, show a distinct laminin basement membrane, and express alpha6beta1 integrins at their basal end. Exposure to conditioned medium from NIH 3T3 cultures accelerates glandular morphogenesis. Parallel cultures maintained as monolayers on plastic remain as monolayers. In the presence of the synthetic androgen mibolerone, acinar cells express prostate specific antigen (PSA) as determined by immunostaining. We conclude that normal prostate cells can undergo acinar morphogenesis while tumorigenic cells have lost this ability. The 3-D cultures provide physiologically relevant in vitro models for elucidating regulation of growth, morphogenesis and differentiation in the normal human prostate, for defining heterotypic interactions in benign prostatic hyperplasia and for establishing the basis for the loss of normal cell organization in early neoplastic lesions such as PIN as well as during tumor progression in prostate cancer.