This paper describes a method for obtaining cultures of rat ventral prostate epithelial cells. The prostate is first perfused with a collagenase solution before removal from the animal; subsequent mincing and incubation in vitro produces a suspension of alveolar cell clumps. Upon incubation, these clumps attach to the surface of the culture dish and spread into discrete epithelial cell colonies, which both retain differentiated morphology, and secrete a species of plasminogen activator that is characteristic of prostatic tissue. These properties were not observed in cultures prepared from single cell suspensions of the same organ. Maintenance of epithelial colony integrity and secretory activity specifically required the continued presence of stromal cells, glucocorticoids and insulin. Androgenic steroids were much less effective than glucocorticoids in stimulating plasminogen activator secretion and in maintaining colony integrity, in spite of the well-established androgen dependence of prostatic tissue morphology in vivo and in organ culture. Furthermore, no effects of prolactin were observed, either when this hormone was tested alone or in conjunction with steroid hormones. Of 3 retinoids tested, retinal was highly cytotoxic at concentrations in the range of 1 microM, whereas retinol and retinoic acid were without detectable effect.