To elucidate the relative importance of pituitary and hypothalamic sites of action of gonadal steroids in modifying gonadotropin secretion, the effects of gonadal insufficiency and of estrogen treatment on the secretion of LH and LH-releasing factor (LRF) were measured in men with carcinoma of the prostate and in male transsexuals. The bioassay of LRF was performed in ovariectomized rats pretreated with estrogen and progesterone, and the change in rat plasma LH from before to 10 min following iv injection of methanol extracts of 40 ml of plasma was the measure of LRF in human plasma. LRF was not detectable in 24 samples from 16 normal men nor in 14 samples from 4 men within 30 days after castration. Men sampled more than 30 days after castration had significantly elevated plasma LRF levels. Plasma LH was significantly elevated within 24 hr after castration in 4 subjects but subsequently declined in one, and then rose again as plasma LRF activity levels increased. Treatment with estrogens for periods of from 7 days to 36 months led to significant increases in plasma LRF, and to significant suppression of plasma LH in all subjects. These observations suggest that the early hypersecretion of LH in castrates is probably due to increased pituitary sensitivity to LRF while later LH secretion is due at least in part to enhanced LRF secretion. Taken together, these findings are indicative of the negative feedback effect of gonadal steroids on both hypothalamic, and pituitary function. On the other hand, enhanced LRF secretion and inhibited LH secretion following long term estrogen treatment suggest a positive feedback effect on the hypothalamus accompanied by reduced pituitary responsiveness to LRF.