It has been previously suggested that the formation of 17β–hydroxy–5α–androstan– 3–one (androstanolone) and 5α–androstane–3β,17β– diol (3β–androstanediol) is important for testosterone action. This observation was extended to androstenedione Explants of ventral prostate glands obtained from normal rats were incubated for 24 hr in the culture medium containing 3H—androstenedione 1.5–3γM, and the radioactive compounds present in the medium and tissue were analyzed. Androstanolone and 5α–androstane–3,17–dione (androstanedione) were the main metabolites. Androsterone, 5α–androstane–3α,17β–diol (androstanediol) and 3β–androstanediol were identified. Testosterone was not found, and therefore androstanolone formation from androstenedione might proceed through androstanedione. In organ cultures grown for 6 days, the activity of androstenedione, androstanedione and testosterone, O.1–35μM, was tested. All three compounds had the same activity at 4.3μM. At lower concentrations, androstanedione was more active, and androstenedione less active than testosterone. Electron microscopic studies showed that 0.3μM testosterone fully maintains the rough endoplasmic reticulum, the Golgi apparatus, the microvilli and the secretory granules of the epithelial cells. Androstenedione has similar effects with the exception that secretory granules were not observed. The results indicate that androstenedione is active in the rat ventral prostate organ culture system and that this might be attributed to the formation of active 5α–reduced metabolites inside the target organ. (Endocrinology91: 396, 1972)