Clomiphene citrate, an “anti-estrogen” with mild estrogenic properties, inhibits rather than stimulates gonadotropin excretion in prepubertal and early pubertal children. These and other data suggest that the sensitivity of the hypothalamic-pituitary “gonadostat” decreases at the onset of puberty. To test this hypothesis further, the daily excretion of urinary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) was determined in 19 children (5 “short normals” and 14 with isolated human growth hormone (HGH) deficiency) who were given ethinyl estradiol (EE) 1.4-14.7 μg/m² per day (2-10 μg/day) for 4 to 7 days. In addition, plasma and urinary gonadotropins and plasma estrogens were serially determined in two prepubertal females(with isolated HGH deficiency) given two injections (24 h apart) of estradiol benzoate, 10 μg/kg. FSH and LH concentrations in plasma and kaolin-acetone urinary concentrates and plasma 17β-estradiol (E₂) and estrone (E₁) were measured by radioimmunoassays. 2-3 μg/m² per day of EE significantly suppressed urinary FSH (and LH when detected in the control period) in two out of six prepubertal children, while all doses >5 μg/m² per day suppressed urinary gonadotropins to undetectable levels in eight prepubertal subjects. In early to midpubertal subjects. 2-10 μg/m² per day of EE produced a slight suppression of urinary FSH, but failed to suppress to undetectable levels. Two subjects in late puberty (stage 4) did not suppress their urinary FSH while on 7 and 8.3 μg/m² per day. In both subjects treated with estradiol benzoate, plasma FSH promptly decreased after the first injection. Urinary FSH was suppressed to <0.1 IU/day on day 2 and urinary and plasma gonadotropins remained suppressed for the duration of the study (3 days). Plasma E₂ and E₁ rose from prepubertal values to peak concentrations of 150 to 250 pg/ml (E₂), and 50 and 100 pg/ml (E₁) at approximately 36 h. We conclude that the hypothalamic-pituitary-gonadal axis is operative in the prepubertal child and that the sensitivity of the hypothalamic-pituitary center(s) which control the secretion of FSH and LH decreases at the onset of puberty in man.