Human prostatic adenocarcinoma fragments (1-6mm) were cultured on collagen sponges in medium supplemented or not supplemented with 4,5alpha-dihydrotesterone (DHT) until 3 weeks, maintaining the three-dimensional (3D) epithelial and stromal organization present in the tumor in vivo. With time, in the presence of DHT, locally progressive cribriform nests of neoplastic cells with proliferative rates higher than those inside the fragment developed on the surface, while the stroma became more dissociated, and fibrosis replaced the muscular component. The 3D-culture provides a promising approach for studying the development and phenotype of prostate epithelial tumor progenitor cells and their stromal interactions.