We have established and characterized two cell lines from the ventral prostate gland of normal Nb rats: NbE-1 (prostatic epithelial) and NbF-1 (prostatic fibroblast) cell lines. To identify the direct mitogenic action of dihydrotestosterone (DHT), we incubated these cell lines alone and together (in the presence and absence of cell contact) with various concentrations of DHT (0.1-10,000 ng/ml) for 24-72 h and assayed for the rate of DNA synthesis and the total number of cells in tissue culture at specified time periods. Results demonstrate that the primary target for DHT mitogenic action is the prostatic fibroblasts. DHT inhibited the growth of prostatic epithelial cells by themselves, but stimulated prostatic epithelial cell growth when the epithelial cells were cocultured with prostatic fibroblasts. Furthermore, the cell-conditioned medium obtained from either the fibroblast or the epithelial cells stimulated in an autocrine or a paracrine manner the growth of prostatic cells in culture. These results are consistent with the concept that DHT stimulates the growth of prostatic epithelial cells indirectly via its direct mitogenic action on the prostatic fibroblasts. Because epithelial cells are the cell type principally responsible for converting testosterone to DHT, and the fibroblasts respond to the mitogenic action of DHT, our results support the concept that tight metabolic cooperation exists between prostatic epithelial and fibroblast cells. These data are in agreement with previous in vivo studies in which we have demonstrated that androgen receptors in the mesenchyme are obligatory for androgen-induced prostate growth and development.