The concentration of free estrogen in 1ml of fractionated plasma obtained from the ovarian venous blood of rats showing a regular 4-day cycle was determined by a bioassay method based on 2, 3, 5-triphenyltetrazolium chloride reduction in the mouse vagina.A significant amount of estrogen, 0.2ng/ml plasma of estradiol equivalent, wasdetected in the ovarian venous blood plasma during diestrus Day 1 and on the morning of diestrus Day 2.The estrogen level markedly elevated in the afternoon of diestrus Day 2 and continued to rise up to a peak (4.5ng/ml) before noon of proestrus.The highly elevated estrogen level sharply declined about 10hrs.prior to ovulation when LH was supposedly released from the pituitary.No significant increase of estrogen level was seen thereafter until diestrus Day 2 of the next cycle.A sharpdecline of ovarian venous blood estrogen to the level of diestrus was induced within 3 hrs.after removal of the pituitary in the afternoon of proestrus when estrogen secretion was highly stimulated.Intravenous injection of gonadotropin, 250μg of NIH-FSH-S₁ or NI.H-LH-S₃, caused a significant stimulation of the ovarian estrogen secretion in the rat hypophysectomized at proestrus.The mechanisms controlling the ovarian estrogen secretion in the rat were discussed.