Technical Abstract: Background: Alcohol ingestion is positively related to breast cancer risk in most epidemiologic studies, but results are heterogeneous at lower levels of intake and the causal nature of the association remains controversial. We performed a controlled feeding study to evaluate the effect of chronic moderate alcohol ingestion on serum levels of hormones that have been related to an increased risk of breast cancer. Methods: Participants included 51 healthy postmenopausal women not using hormone replacement therapy. Each participant consumed 15 gm alcohol/day, 30 gm alcohol/day, or a placebo beverage during one of three 8-week dietary periods. The order of assignment to the three alcohol levels was random. Each dietary period was preceded by a 2-5 week washout period. All food and beverages were supplied by the study during the dietary periods, and energy intake was adjusted to keep body weight constant. Estradiol, estrone, estrone sulfate, testosterone, androstenedione, progesterone, dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenediol were measured in serum collected at the end of each dietary period. Results: When consuming 15 gm and 30 gm alcohol/day, respectively, participants' estrone sulfate concentrations increased by 7.5% (95% confidence interval (CI) = -0.3, 15.9) and 10.7% (95% CI = 2.7, 19.3), and their DHEAS concentrations increased by 5.1% (95% CI = 1.4, 9.0) and 7.5% (95% CI = 3.7, 11.5). None of the other hormones measured changed significantly when women consumed alcohol. Conclusions: Results from this study suggest a mechanism by which consumption of 1-2 alcoholic drinks/day by postmenopausal women could increase their risk of breast cancer.