Breast cancer risk increases with increased levels of alcohol consumption, potentially through an effect on sex hormone levels. In a cross-sectional study among Dutch participants (n = 17,357) of the European Prospective Investigation into Cancer and Nutrition conducted in Utrecht, The Netherlands (Prospect-EPIC), we investigated the relation between alcohol intake and estrogen and androgen levels. Alcohol intake was calculated from a food frequency questionnaire. Women were included if they were postmenopausal, had donated a blood sample, and did not use hormone replacement therapy or oral contraceptives at the time of blood donation (n = 1093). Women who consumed more than 25 g of alcohol per day had higher levels of estrone (P(trend) = 0.001), estradiol (P(trend) = 0.03), dehydroepiandrosterone sulfate (P(trend) = 0.18), and higher estrone/estradiol (P(trend) = 0.14) and estrone/androstenedione (P(trend) = 0.06) ratios, compared with nondrinkers. Levels of androstenedione, testosterone, and SHBG did not differ between women who consumed alcohol and nondrinkers. Furthermore, there were no differences in the free androgen index or estradiol to testosterone ratio. In conclusion, levels of estrogens and dehydroepiandrosterone sulfate are higher in women who consume more alcohol. This finding supports the hypothesis that alcohol use may increase breast cancer risk at least partially through an effect on sex steroid levels.