Several lines of evidence suggest that vitamin D may reduce incidence of breast cancer, but few epidemiologic studies have addressed the relation of plasma vitamin D metabolites to the risk of this disease. We prospectively examined the relationship between plasma levels of 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] and risk of breast cancer in a case-control study nested within the Nurses' Health Study cohort. Blood samples were collected from study participants in 1989-1990. Breast cancer cases developing between blood collection and June 1, 1996, were matched to cancer-free controls on the basis of age, menopausal status, and other factors. Stored plasma samples from 701 cases and 724 controls were available for metabolite analysis. Cases had a lower mean 25(OH)D level than controls (P = 0.01), but mean 1,25(OH)2D levels were similar (P = 0.49). High levels of both metabolites were associated with a nonsignificant lower risk of breast cancer. Women in the highest quintile of 25(OH)D had a relative risk of 0.73 (95% confidence interval = 0.49-1.07; Ptrend = 0.06) compared with those in the lowest quintile. For 1,25(OH)2D, the comparable relative risk was 0.76 (95% confidence interval = 0.52-1.11; Ptrend = 0.39). For both metabolites, the association was stronger in women ages 60 years and older, but results were not statistically significant. Our findings suggest that high levels of 25(OH)D, and perhaps 1,25(OH)2D, may be modestly associated with reduced risk of breast cancer.