Endogenous sex hormones play an important role in the etiology of breast cancer. Polymorphisms in genes encoding for enzymes involved in steroidogenesis may therefore play a role in breast cancer risk. Cytochrome P450c17alpha (Cyp17) functions at key branch points in human steroidogenesis. A T-->C transition (A1 and A2 allele) in the 5' untranslated region may be associated with increased expression of Cyp17. Using a case-cohort design, we studied the effects of the A2 allele on endogenous sex hormone levels and breast cancer risk within a large population-based cohort (n = 9,349) in the Netherlands (the DOM-cohort). Cyp17 genotype was determined in 335 incident postmenopausal breast cancer cases, which occurred after follow-up (median time to follow-up, 19 years) of the entire cohort, and in a random sample of 373 women (subcohort). Concentrations of estrone (E1), estradiol (E2), testosterone, 5alpha-androstane-3alpha, 17beta-diol (3alphaD), and creatinine were measured in first-morning urine samples. Only among women with body mass index (BMI) < 25 kg/m2 was the A2A2 genotype associated with higher levels of E1, E2, and 3alphaD compared with a group of women with either the A1A1 or the A1A2 genotype (e.g., geometric means of E(1) in ng/mg(creatinine): A2A2, 2.23; A1A1/A1A2, 1.47; P = 0.03). Adjusted breast cancer rate ratios for women with the A1A2 or A2A2 genotype compared with women with the A1A1 genotype were 0.96 (0.68-1.37) and 0.80 (0.47-1.35), respectively. These results did not differ between women with low and high BMI. In conclusion, this paper shows that women with low BMI and the A2A2 genotype had higher endogenous sex steroid levels compared with women with the A1A1 genotype. However, these increased sex steroid levels are not translated into an increased breast cancer risk in these women.