Testes of 28- and 70-day-old golden hamsters were separated into seminiferus tubules and interstitial tissue by wet dissection. Homogenates of the tissues and subcellular fractions were incubated with [14C]androstenedione, and NADPH and enzyme activity (nanomoles per 100 mg protein/h) was estimated. In immature testes, the 5 beta-reductase activity was largely confined to the seminiferous tubules, while 5 alpha-reductase was localized in the interstitial tissue. The activity of 17 beta-ol-dehydrogenase was found to be equally distributed between tubules and interstitial tissue. Although the activities of 5 beta- and 5 alpha-reductases were approximately 10 times greater in immature than adult testes, the 17 beta-ol-dehydrogenase activity in adult testes was approximately 10 times that in immature testes. In the immature testes, most 5 alpha-reductase and 17 beta-ol-dehydrogenase were localized in microsomes, whereas 5 beta-reductase was wholly localized in cytosol. These results indicates that 5 beta-reduction of delta 4-3-ketosteroids takes place largely in cytosol of tubular cells, while 5 alpha-reduction occurs in microsomes of interstitial cells in the testis of the immature golden hamster.