Previous studies have shown that progesterone concentrations in serum and nipple aspirate fluid (NAF) are significantly correlated in premenopausal women, but estradiol concentrations are not. We therefore sought to ascertain the patterns of both steroids in NAF throughout the menstrual cycle and in postmenopausal women. Simultaneous samples of blood and NAF were obtained from 40 premenopausal and 16 postmenopausal women. Premenopausal samples were backdated from the following menstrual period. Steroids were purified by high-performance liquid chromatography before quantification by immunoassays. Serum steroids and NAF progesterone followed the expected pattern across the menstrual cycle, with a midcycle peak of estradiol and a midluteal peak of progesterone. However, the estradiol peak in NAF occurred about a week after the serum peak in the midluteal phase, when serum estradiol had declined to less than half the value at midcycle. NAF estrone was also elevated at the midluteal phase. Potential estrogen precursors androstenedione, estrone sulfate, and dehydroepiandrosterone sulfate declined in NAF from midcycle to the midluteal phase as NAF estradiol was increasing. Progesterone concentrations were significantly lower in NAF in postmenopausal women than in premenopausal women, but estrogen concentrations were not. This is the first description of the temporal relationships of sex steroids in NAF and serum relative to the menstrual cycle. These results provide insights into the lack of correlation of NAF and breast tissue estrogens with serum estrogens, and generate new hypotheses.