Oxytocin is present in the male reproductive tract where it is known to modulate contractility, cell growth and steroidogenesis. Little is known about how oxytocin regulates these processes. This study describes the localization of oxytocin receptor in the rat ventral prostate and investigates if oxytocin regulates gene expression and/or activity of 5 -reductase isoforms I and II. The ventral prostates of adult male Wistar rats were collected following daily subcutaneous administration of saline (control), oxytocin, a specific oxytocin antagonist or both oxytocin plus antagonist for 3 days. Expression of the oxytocin receptor was identified in the ventral prostate by RT-PCR and Western blot, and confirmed to be a single active binding site by radio-receptor assay. Immunohistochemistry localized the receptor to the epithelium of prostatic acini and to the stromal tissue. Real time RT-PCR determined that oxytocin treatment significantly reduced expression of 5 -reductase I, but significantly increased 5 -reductase II expression in the ventral prostate. Activity of both isoforms of 5 -reductase was significantly increased by oxytocin resulting in increased concentration of prostatic dihydrotestosterone. In conclusion, oxytocin is involved in regulating conversion of testosterone to the biologically active dihydrotestosterone in the rat ventral prostate. It does so by differential regulation of 5 -reductase isoforms I and II.