A sensitive binding assay for estradiol has been developed by the use of pregnancy plasma. The binding protein was partially purified by gel filtration and separation of bound and free tracer estradiol was performed by charcoal adsorption. Plasma extracts were purified by Sephadex adsorption prior to assay, giving complete separation from neutral steroids with an estradiol recovery of 53%. The blank of the method was 0.4 ng/100 ml, and the betweenassay reproducibility was ±10%. The mean plasma estradiol concentration was 2.5 ng/100 ml in postmenopausal females, and 2.4 ng/100 ml in males. The plasma levels of estradiol during the menstrual cycle were: 1–10 ng/100 ml in the early follicular phase, rising to 14–77 ng/100 ml and falling again to lower levels (3–14 ng/100 ml) at the time of the midcycle LH peak. During the luteal phase, plasma estradiol rose again to 19–34 ng/100 ml in most subjects. The temporal relationship between plasma estradiol and LH levels is consistent with the proposal that the ovulatory LH surge is stimulated by a preceding peak of plasma estradiol. The lowest levels of plasma estradiol were observed at the time of menstruation, and during oral contraceptive therapy.