Abstract Female rats were hypophysectomized at 21 days of age, and after a lapse of 3 days the hypophysectomized rats in groups of 4–13 were injected daily with NIH-LH-S19, NIAMD-Rat-FSH-B-I, 5α-androstane-3α, 17β-diol, testosterone, oestradiol-17β, saline or sesame oil for 3 days. Ovarian homogenates from these rats at 27 days of age were incubated with [ 14C]-testosterone and 5α-reductase activities were estimated. The 5α-reductase activity (nmol/g tissue/h or nmol/both ovaries/h) decreased significantly 6 days following hypophysectomy. A distinct response to LH in 5α-reductase activity of the hypophysectomized rat ovaries was found, with a dose-response in activity from 260 ± 94 (S.D.) in the hypophysectomized control to the maximum level of 1960 ± 177 (S.D.) nmol/g/h, using doses from 3 to 90 μg per day. In contrast, FSH was not effective, for it had little effect on 5α-reductase activity even when 10 or 50 μg was given each day. No stimulation of 5α-reductase activity by injection of androgens and oestradiol-17β in large doses was involved, showing that the effect of LH is not mediated by sex steroids. These results show that 5α-reductase activity in prepubertal rat ovaries is regulated by LH.