One of the mechanisms through which advanced prostate cancer (PCa) usually relapses after androgen deprivation therapy (ADT) is the adaptation to residual androgens in PCa tissue. It has been observed that androgen biosynthesis in PCa tissue plays an important role in this adaptation. In the present study, we investigated how stromal cells affect adrenal androgen dehydroepiandrosterone (DHEA) metabolism in androgen-sensitive PCa LNCaP cells. DHEA alone had little effect on prostate-specific antigen (PSA) promoter activity and the proliferation of LNCaP cells. However, the addition of prostate stromal cells or PCa-derived stromal cells (PCaSC) increased DHEA-induced PSA promoter activity via androgen receptor activation in the LNCaP cells. Moreover, PCaSC stimulated the proliferation of LNCaP cells under physiological concentrations of DHEA. Biosynthesis of testosterone or dihydrotestosterone from DHEA in stromal cells and LNCaP cells was involved in this stimulation of LNCaP cell proliferation. Androgen biosynthesis from DHEA depended upon the activity of various steroidogenic enzymes present in stromal cells. Finally, the dual 5α-reductase inhibitor dutasteride appears to function not only as a 5α-reductase inhibitor but also as a 3β-hydroxysteroid dehydrogenase inhibitor in LNCaP cells. Taken together, this coculture assay system provides new insights of coordinate androgen biosynthesis under the microenvironment of PCa cells before and after ADT, and offers a model system for the identification of important steroidogenic enzymes involved in PCa progression and for the development of the corresponding inhibitors of androgen biosynthesis.