5 alpha-Reductase and aromatase activities were measured in fetal and newborn rat gubernaculum and other tissues by radiometric assays using [1 beta-3H]testosterone as substrate. Aromatase activity was measured by the stereospecific release of tritium into the incubation medium, and 5 alpha-reductase was assessed by measuring the amount of 5 alpha-reduced steroids produced during the same incubations. Aromatase activity was low (less than 0.2 pmol/h.mg protein) in gubernaculum at all ages studied, whereas the activity in postnatal ovary ranged from 3.9-20 pmol/h.mg protein. 5 alpha-Reductase activity was relatively high in day 18 fetal gubernaculum (approximately 115 pmol/h.mg protein), but 2 days later in development (fetal day 20), 5 alpha-reductase activity had declined to approximately 21 pmol/h.mg protein. 5 alpha-Reductase activity was very low (less than 10 pmol/h.mg protein) in the postnatal gubernaculum. Histological examination of day 18 fetal gubernaculum indicated that it is composed of dense, poorly organized, mesenchymal tissue. By day 20 of gestational development, primitive muscle cells are recognizable in the periphery of the gubernaculum, and by day 3 of postnatal development the gubernaculum is composed almost entirely of muscle. These findings suggest that 5 alpha-reductase activity may be located in the mesenchymal cells and may be important in early differentiation of the gubernaculum in the male. The administration of a 5 alpha-reductase inhibitor to pregnant rats from days 14-22 of gestation inhibited the normal growth rate of the gubernaculum, as assessed by measuring the protein content of the gubernaculum in control and treated rats, but did not have any profound effect on the histological development of the gubernaculum.