Estrogens exert their effect on the breast through the estrogen receptor. We prospectively investigated breast cancer risk associated with 2 polymorphic sites in the estrogen receptor alpha gene (ESR1). A total of 4,248 Caucasian women from the Study of Osteoporotic Fractures were genotyped for the 2401 T/C and 2354 A/G polymorphisms in ESR1. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the associations between genotypes and breast cancer. During a mean follow-up of 12.4 years, 252 (5.9%) women developed breast cancer. The HR (95% CI) for breast cancer were 0.928 (0.708, 1.22) and 0.834 (0.538, 1.29) for the 2354 A/ G and A/A genotypes, respectively. Interactions with 2354 variant were observed for smoking (HR 5 1.52 and 1.56 for A/G and A/A smokers, respectively; HR 5 0.74 and 0.60 for A/G and A/A nonsmokers, respectively; interaction p 5 0.03) and walking (HR 5 0.75 and 1.15 for A/G and A/A walkers, respectively; HR 5 0.18 and 0.49 for A/G and A/A non-walkers, respectively; interaction p 5 0.01). There were no differences in the HR for the 2401 T/C genotypes. An interaction between parity and carriage of the T allele was found (HR 5 0.60 vs. 1.12 for nulliparous vs. parous women; interaction p 5 0.03). ESR1 polymorphisms in combination with lifestyle factors may be associated with breast cancer risk in older Caucasian women. ' 2005 Wiley-Liss, Inc.Key words: breast neoplasms; estrogen receptor; genetic polymorphisms; epidemiology; prospective cohort study The actions of estrogen, believed to be a causal factor in postmenopausal breast cancer, 1-4 are mediated by the estrogen receptors (ER). The ER has 2 major forms, a and b. 5,6 The ER-a (ESR1) is of interest in breast cancer because its expression has been associated with the disease. In particular, breast cancer occurs more often when ESR1 is over-expressed in adjacent normal epithelium. 7 Moreover, increased ESR1 expression in normal epithelium is found in older women, 8 as well as in Caucasian women compared to non-Caucasian 9 and Asian 8 women, reflecting age and ethnic-associated patterns of breast cancer risk. In addition, estrogen receptor status is a prognostic factor in breast cancer and a strong predictor of response to endocrine therapy. 10 The ESR1 gene contains several single nucleotide polymorphisms (SNP) 8,11-14 whose functional significance remains unknown. Nonetheless, 2 of these SNP located in intron 1, the IVS1 2401 and IVS1 2354 A/G variants, have been associated with hormonally-related diseases including breast, [15][16][17][18][19] prostate 20 and endometrial 21 cancers. These same 2 SNP have also been associated with other estrogen-linked conditions, 22,23 as well as factors associated with increased breast cancer risk, such as age at menopause 24 and age at menarche. 25 In addition, increased bone mineral density, a potential surrogate marker of cumulative estrogen exposure and a predictor of breast cancer risk, 26 has also been associated with these SNP, 27,28 although...