Experimental and epidemiologic studies suggest that vitamin D metabolites (1,25-dihydroxyvitamin D [1,25(OH)(2)D] and its precursor 25-hydroxyvitamin D [25(OH)D]) may reduce breast cancer risk. We examined subsequent breast cancer risk related to serum levels of these metabolites. In a cohort of women ages 55 to 74 years, who donated blood at baseline (1993-2001) in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we identified 1,005 incident breast cancer cases during follow-up through 2005 (mean time between blood draw and diagnosis, 3.9 years). Noncases (n = 1,005) were frequency matched to the cases based on age and year of entry. Sample weights that accounted for unequal probabilities of selecting cases and noncases were applied to make inferences that reflected the entire Prostate, Lung, Colorectal, and Ovarian cohort. Using Cox proportional hazards modeling, we computed breast cancer relative risks (RR) and 95% confidence intervals (95% CI) by quintile for each metabolite. The RR of breast cancer for the highest quintile of 25(OH)D concentration versus the lowest was 1.04 (95% CI, 0.75-1.45; P(trend) = 0.81). Similarly, the breast cancer RR for the highest quintile of 1,25(OH)(2)D compared with the lowest was 1.23 (95% CI, 0.91-1.68; P(trend) = 0.14). Excluding the first 2 years of follow-up did not materially alter these estimates. There was also no evidence of inverse risk in older women (> or =60 years) versus younger women (<60 years). In this prospective study of postmenopausal women, we did not observe an inverse association between circulating 25(OH)D or 1,25(OH)(2)D and breast cancer risk, although we cannot exclude an association in younger women or with long-term or earlier exposure.