The role of follicle-stimulating hormone (FSH) in the regulation of estrogen biosynthesis in vivo has been investigated in immature hypophysectomized rats, utilizing uterine weight and histologic responses, and ovarian estradiol-17beta concentrations as indicators of estrogen secretion. A highly purified FSH preparation produced only borderline increases in uterine weights and in ovarian estradiol-17beta contents when administered alone at 2.5 mug/day for 3 days. Testosterone or androstenedione (5 mg/day), when administered in the absence of FSH, produced typically androgenic stimulation of uteri, and did not increase ovarian estradiol-17beta concentrations. When administered concomitantly with FSH, the uterine weight-stimulating activity of these aromatizable androgens was substantially increased, accompanied by marked hypertrophy of the endometrial mucosal cells, and ovarian estradiol-17beta concentrations were increased 20- to 200-fold. The administration of the non-aromatizable androgen, 17beta-OH-5alpha-androstan-3-one (DHT) (5 mg/day, by itself produced uterine weight increases similar to those caused by testosterone alone; however, no evidence of increased estrogen secretion resulted from the combined treatment of DHT wtih FSH. A highly purified luteinizing hormone (LH) preparation was equally as effective as exogenous androstenedione in increasing ovarian concentrations of immunoreactive androgen (testosterone + DHT) but evoked none of the above signs of estrogen secretion unless administered together with FSH. The weights of ovaries were not affected by the administration of LH or of any of the androgens by themselves, but were approximately doubled by FSH alone. Ovarian weights were increased still further when FSH was administered concomitantly with LH, testosterone , or androstenedione, but not with DHT. It is concluded that FSH and LH regulate ovarian estrogen secretion in vivo by acting at biochemically distinct sites--LH stimulating the synthesis of C19-steroids, which are then converted to estradiol-17beta under specific stimulation by FSH.