Abstract Background: Epidemiological studies provide strong evidence for a role of endogenous sex steroids in the etiology of breast cancer. Our aim was to identify common variants in genes involved in sex steroid synthesis or metabolism that were associated with premenopausal hormone levels and breast cancer risk. Methods: We measured urinary estrone glucuronide (E1G) and pregnanediol glucuronide (PG) using a protocol specifically developed to account for cyclic variation in hormone levels during the menstrual cycle, plasma sex hormone-binding globulin (SHBG) and androgenic precursors in up to 763 healthy premenopausal women. We genotyped 642 single nucleotide polymorphisms (SNPs) in these women; a single SNP was further tested for association with breast cancer risk in data from 10,551 breast cancer case patients and 17,535 control subjects. All statistical tests were two-sided. Results: rs10273424 mapping approximately 50kb centromeric to the cytochrome P450 3A (CYP3A) cluster (7q22.1) was associated with a 21.8% reduction in E1G levels (P = 2.7 × 10−9) and a modest reduction in breast cancer risk in cases diagnosed at or before age 50 (OR = 0.91; P = 0.03) but not older cases (odds ratio (OR) = 1.01; P = 0.82). A rare non-synonymous SHBG SNP was associated with reduced plasma SHBG levels. Conclusions: Genetic variation in non-coding sequences flanking the CYP3A locus contributes to variance in premenopausal E1G levels and breast cancer risk in younger cases. Since CYP3A4, the most predominantly expressed CYP3A gene, is responsible for metabolism of endogenous and exogenous hormones and hormonal agents such as tamoxifen, used in the treatment of breast cancer this association may have wider implications. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-08-04.