Contrasting etiologic hypotheses about the role of endogenous sex steroids in breast cancer development among premenopausal women implicate ovarian androgen excess and progesterone deficiency, estrogen excess, estrogen and progesterone excess, and both an excess or lack of adrenal androgens (dehydroepiandrosterone [DHEA] or its sulfate [DHEAS]) as risk factors. We conducted a case-control study nested within the European Prospective Investigation into Cancer and Nutrition cohort to examine associations among premenopausal serum concentrations of sex steroids and subsequent breast cancer risk. Levels of DHEAS, (Delta4-)androstenedione, testosterone, and sex hormone binding globulin (SHBG) were measured in single prediagnostic serum samples from 370 premenopausal women who subsequently developed breast cancer (case patients) and from 726 matched cancer-free control subjects. Levels of progesterone, estrone, and estradiol were also measured for the 285 case patients and 555 matched control subjects who had provided information about the day of menstrual cycle at blood donation. Conditional logistic regression models were used to estimate relative risks of breast cancer by quartiles of hormone concentrations. All statistical tests were two-sided. Increased risks of breast cancer were associated with elevated serum concentrations of testosterone (odds ratio [OR] for highest versus lowest quartile = 1.73, 95% confidence interval [CI] = 1.16 to 2.57; P(trend) = .01), androstenedione (OR for highest versus lowest quartile = 1.56, 95% CI = 1.05 to 2.32; P(trend) = .01), and DHEAS (OR for highest versus lowest quartile = 1.48, 95% CI = 1.02 to 2.14; P(trend) = .10) but not SHBG. Elevated serum progesterone concentrations were associated with a statistically significant reduction in breast cancer risk (OR for highest versus lowest quartile = 0.61, 95% CI = 0.38 to 0.98; P(trend) = .06). The absolute risk of breast cancer for women younger than 40 followed up for 10 years was estimated at 2.6% for those in the highest quartile of serum testosterone versus 1.5% for those in the lowest quartile; for the highest and lowest quartiles of progesterone, these estimates were 1.7% and 2.6%, respectively. Breast cancer risk was not statistically significantly associated with serum levels of the other hormones. Our results support the hypothesis that elevated blood concentrations of androgens are associated with an increased risk of breast cancer in premenopausal women.