To investigate the effects of MK906, a selective 5 alpha reductase (5αR) type 2 (5αR2) inhibitor, and of MK386, a specific 5αR1 inhibitor, on the cellular proliferation of androgen-dependent human prostatic cancer (PCa) cells in cultures of cells derived from bioptic and surgical tissues. In this study we tested the effects of MK906 and MK386 in 30 cultures derived from PCa, 6 from PIN and 10 from benign prostatic hyperplasia specimens. Prostate primary cultures under short-term conditions (with <4 subcultures) represent a mixture of epithelial and stromal cells. Epithelial cells require testosterone (T) for optimal growth, but were not able to grow in the presence of T under long-term conditions even if DHT was able to induce cellular proliferation to a similar extent in both conditions, suggesting that 5αR can be lost in long-term cultures. Therefore, our studies were performed under short-term conditions. Both 5αR inhibitors decreased cell proliferation significantly and dose-dependently in all the samples tested. MK906 was more efficient than MK386 in 7 out of 10 cultures derived from BPH tissues, in 4 out of 6 cultures derived from PIN and in 18 out of 30 cultures derived from PCa. In 3 out of 10 BPH, in 2 out of 6 PIN and in 5 out of 30 PCa-derived cultures, both inhibitors presented similar efficacy, whereas in 1 out of 10 BPH and 7 out of 30 PCa-derived cultures MK386 was more efficient than MK906. In addition, MK386 was more efficient than MK906 in 4 out of 15 non-metastatic PCa and 2 out of 7 metastatic PCa-derived cultures. Considering that 5αR1 (responsible primarily for androgenic catabolism) is mostly expressed in epithelial cells and that 5αR2 (responsible for local DHT synthesis and release) is expressed in the stromal cells (which provides several paracrine growth factors and DHT itself to the epithelial cells), our experiments suggest that the inhibition of both 5αR1 and 5αR2 by MK386 and MK906, respectively, may have therapeutic potential in order to reduce the growth and progression of human prostatic cancers, through the inhibition of autocrine or paracrine mechanisms involving the stromal cell compartment. In addition, some effects of 5αR inhibitors could be mediated by estrogens, which are synthesized by the aromatase enzyme present in the epithelial cells. These aspects could be considered in order to improve the therapeutical management of PCa and for future clinical trials.