Abstract Background: In the Women's Health Initiative Hormone Trials (WHI-HT), breast cancer risk was increased with estrogen plus progestin (E+P) but not with unopposed estrogen (E-alone). We hypothesized that E+P would preferentially metabolize to 16α-hydroxyestrone (16α-OHE1) rather than 2-hydroxyestrone (2-OHE1), and that breast cancer risk would be associated with baseline and 1 year changes in estrogen metabolites: positively for 16α-OHE1 levels and negatively for levels of 2-OHE-1 and the 2:16 ratio. Methods: In a prospective case-control study nested in the WHI-HT, 845 confirmed breast cancer cases were matched to 1,690 controls by age and ethnicity. Using stored serum, 2-OHE1 and 16α-OHE1 levels were measured by enzyme immunoassay at baseline, and for those randomized to active treatment (n = 1,259), at 1 year. Results: The 1-year increase in 16α-OHE1 was greater with E+P than E-alone (median 55.5 pg/mL vs. 43.5 pg/mL, P < 0.001), but both increased 2-OHE1 by ∼300 pg/mL. Breast cancer risk was modestly associated with higher baseline levels of 2-OHE1 and the 2:16 ratio, and for estrogen receptor+/progesterone+ cases only, higher baseline 16α-OHE1 levels. For those randomized to active treatment, breast cancer risk was associated with greater increase in 2-OHE-1 and the 2:16 ratio, but associations were not significant. Conclusions: Although E+P modestly increased 16α-OHE1 more than E-alone, increase in 16α-OHE1 was not associated with breast cancer. Impact: Study results do not explain differences between the WHI E+P and WHI E-alone breast cancer results but metabolism of oral HT, which may explain smaller than expected increase in breast cancer compared with endogenous estrogens. Cancer Epidemiol Biomarkers Prev; 21(11); 2022–32. ©2012 AACR.