BACKGROUND: Prospective studies have examined the association of serum and plasma carotenoids and micronutrients and breast cancer; however, to date, studies have only assessed exposure at one point in time. OBJECTIVE: This study analyzed baseline and repeated serum measurements of carotenoids, retinol, and tocopherols to assess their associations with postmenopausal breast cancer risk. DESIGN: Serum concentrations of alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene, lutein + zeaxanthin, retinol, alpha-tocopherol, and gamma-tocopherol were measured in a 6% sample of women in the Women's Health Initiative clinical trials at baseline and at years 1, 3, and 6 and in a 1% sample of women in the observational study at baseline and at year 3. The association of baseline compounds and breast cancer risk was estimated by Cox proportional hazards models. In addition, repeated measurements were analyzed as time-dependent covariates. Of 5450 women with baseline measurements, 190 incident cases of breast cancer were ascertained over a median of 8.0 y of follow-up. RESULTS: After multivariable adjustment, risk of invasive breast cancer was inversely associated with baseline serum alpha-carotene concentrations (hazard ratio for highest compared with the lowest tertile: 0.55; 95% CI: 0.34, 0.90; P = 0.02) and positively associated with baseline lycopene (hazard ratio: 1.47; 95% CI: 0.98, 2.22; P = 0.06). Analysis of repeated measurements indicated that alpha-carotene and beta-carotene were inversely associated with breast cancer and that gamma-tocopherol was associated with increased risk. CONCLUSIONS: The present study, which was the first to assess repeated measurements of serum carotenoids and micronutrients in relation to breast cancer, adds to the evidence of an inverse association of specific carotenoids with breast cancer. The positive associations observed for lycopene and gamma-tocopherol require confirmation. This trial was registered at ClinicalTrials.gov as NCT00000611.