Background: Mammographic density is one of the strongest predictors of breast cancer risk. The mechanism by which breast density increases breast cancer risk is unclear although it has been hypothesized that breast density reflects cumulative exposure to estrogens. Methods: To evaluate this hypothesis, we conducted a cross-sectional study among 520 postmenopausal women in the Nurses' Health Study that examined the relation between circulating sex hormones and mammographic density. Women were postmenopausal and not taking exogenous hormones at the time of blood collection and mammogram. Percent breast density was measured from digitized mammograms using a computer-assisted method. Circulating estrone, estradiol, androstenedione, testosterone, DHEA, DHEA sulfate, sex hormone-binding globulin, progesterone, and prolactin were measured in plasma. Results: In contrast to the prior hypothesis, circulating estrogens were inversely related to percent mammographic density. The mean percent mammographic density was 25.6% among women in the lowest quartile of circulating estradiol compared with 14.4% among women in the highest quartile [Spearman correlation (r) = -0.22, P < 0.0001]. Circulating estrogens alone explained 1% to 5% of the variation of mammographic density. Body mass index was positively associated with circulating estradiol levels (r = 0.45, P < 0.0001) and inversely related to percent mammographic density (r = -0.51, P < 0.0001). After adjustment for body mass index, there was no association between estradiol and breast density (r = 0.01, P = 0.81). Likewise, there was no relation between the other sex hormones measured or prolactin and mammographic density after adjustment for body mass index. Conclusion: These findings indicate that in postmenopausal women, mammographic density is independent of circulating sex hormone levels.