The neurohypophysis of the spayed femalerat previously injected with a single dose of <sup>3</sup>H-estradiol took up and 75 min later retained significantly more radioactivity than the adenohypophysis. 86 to 92 % of the radioactivity in the neurohypophysis was chromato-graphically identified as estradiol. Cytosol fractions of rat neurohypophysis incubated with <sup>3</sup>H-estradiol and subjected to Sephadex G-25 chromatography exhibited significant amounts of radioactivity bound to high molecular weight components. Treatment with dextran-coated charcoal of nuclei-free neurohypophysial homogenates incubated with <sup>3</sup>H-estradiol of different specific activities uncovered a high affinity, low cupacity binding of estradiol to cytosol components. Testosterone and progesterone were about 60% as active as unlabeled estradiol in causing displacement of <sup>3</sup>H-estradiol from high affinity binding sites. Estradiol uptake by the rat neurohypophysis exhibited significant in vivo 24-h variations, being maximal during day-time. Pinealectomy or superior cervical ganglionectomy caused increases in the uptake of estradiol by the neurohypophysis, which were partially reversed by the administration of melatonin.