A radioimmunoassay for the measurement of estradiol in human milk is evaluated. The detection limit was found to be 25 pg. of estradiol per milliliter of milk. In milk samples collected from four lactating women during three to four months and from one pregnant and lactating woman, the concentration of estradiol was found to be below the detection limit of the assay. When six lactating women were given vaginal suppositories containing 50 or 100 mg. of estradiol, it was possible to estimate the estradiol concentration in milk. A ratio of transfer of estradiol from plasma to milk during physiologic conditions is calculated to be less than 100:10.