Several techniques for the separation of rat ventral prostate cells, using density gradient centrifugation or mechanical means have been published, yielding fibroblast and epithelial cell populations of varying purities and viability. These techniques are often tedious, yield relatively limited numbers of cells, demand considerable technical expertise, and result in the isolation of cells of limited viability. Two techniques for the isolation and establishment of epithelial and fibroblast cell cultures from mature rat ventral prostate are described here. Collagenase/trypsin digestion of the tissue yields a single-cell suspension of both cell types that are separated on Percoll isopycnic centrifugation gradients. A continuous gradient system allows for the separation of a greater number of cells with very high degrees of purity. A second technique based on a step-gradient system produces reproducible subfractionation of the epithelial cell component of the prostate within a considerable shorter period of time. An improved medium for plating epithelial and fibroblast cells has also been developed. The separated cells are plated on collagen and/or fibronectin-coated dishes in a serum-free plating medium that is later replaced with a serum containing growth medium. The plating medium greatly increases the plating efficiency of the isolated cell types, particularly the epithelial cells.