In order to ascertain which ovarian cell type within the follicle is the source of preovulatory estrogen secretion in vivo, ovarian venous, as well as peripheral venous, blood was collected prior to, 5 min, 30 min, and 120 min after the removal of follicular fluid and granulosa cells from 17 monkeys. In addition, estrogen, progesterone, and progestins were measured in the peripheral blood, ovarian venous blood, and follicular fluid of the follicle-containing and contralateral ovary in 24 monkeys, in order to prove that the preovulatory follicle is the principal source of estrogen. Estradiol was the principal estrogen and was secreted in larger amounts by the ovary with the large preovulatory follicle (7-10 mm in diameter) compared with the contralateral ovary. In 15 experiments ovarian venous estrogen (3934 +/- 798 pg/ml, mean +/- SE) in the vein draining the large follicle-containing ovary was usually 5-15-fold higher than peripheral plasma estrogen levels which were 307 +/- 31 pg/ml. The contralateral ovary secreted a small amount of estrogen (654 +/- 162 pg/ml). Follicular fluid contained large amounts of estrogen (2754 +/- 695 ng/ml) with levels which did not always correlate well with peripheral plasma or ovarian venous estrogen. Ovaries containing non-preovulatory or recently ovulated follicles secreted less estrogen. The removal of granulosa cells and follicular fluid from the preovulatory follicle led to no significant decrease (P greater than 0.5) in ovarian venous secretion of estrogen after a 5, 30, or 120 min time interval. This would indicate that, within the time constraints of this experiments, the follicular fluid and granulosa cells contribute relatively little to ovarian venous estrogen and that thecal cells of the large preovulatory follicle alone can secrete more of the estrogen into the ovarian vein.