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Women who metabolize a large proportion of their estrogen via the 16α hydroxylation pathway could be at a higher risk of breast cancer. The objective of this study was to test the hypothesis that serum concentrations of 2-hydroxyestrone (2-OHE1) and 16α-hydroxyestrone (16α-OHE1), as well as their ratio, predict the risk of breast cancer in older women.We performed a case-cohort study of 272 women with confirmed incident breast cancer and 291 controls chosen randomly from the cohort. Estrogen metabolites were measured in serum collected at the baseline examination and stored at −120°C. Incident breast cancers were confirmed by medical records and pathology reports during an average follow-up of 8.7 years.Mean concentrations of 2-OHE1 and 16α-OHE1, adjusted for age and body mass index, were 3% to 4% higher in cases compared with controls: 2-OHE1 was 176 pg/mL and 169 pg/mL and 16α-OHE1 was 233 pg/mL and 226 pg/mL in cases and controls, respectively. There was, however, no difference in the ratio of 2-OHE1 to 16α-OHE1. The risk of breast cancer in women with the highest quartile of this ratio compared with those in the lowest quartile was 1.17 (95% confidence interval = 0.73–1.87).The study results do not support the hypothesis that the ratio of 2-OHE1 to 16α-OHE1 predicts breast cancer risk.