Few modifiable factors are known to reduce ovarian cancer risk. Ecologic studies and experimental data suggest that vitamin D may reduce ovarian cancer risk. Therefore, we examined whether plasma concentrations of 25-hydroxyvitamin D (a measure of overall vitamin D status) and 1,25-dihydroxyvitamin D (biologically active form) were associated with risk of epithelial ovarian cancer in a nested-case control study using data from three prospective cohorts: the Nurses' Health Study (NHS), NHSII, and the Women's Health Study (WHS). The analysis had 224 cases (161 from NHS/NHSII and 63 from WHS) and 603 controls (matching ratio, 1:3 for NHS/NHSII and 1:2 for WHS). Women ranged in age from 34 to 73 years (mean, 56 years). We did not observe significant associations between 25-hydroxyvitamin D [top versus bottom quartile: relative risk (RR), 0.83; 95% confidence interval (95% CI), 0.49-1.39; P(trend) = 0.57] or 1,25-dihydroxyvitamin D levels (RR, 1.14; 95% CI, 0.70-1.85, P(trend) = 0.93) and ovarian cancer risk. Study-specific associations were not statistically significant and no statistical heterogeneity existed between studies (P = 0.66, 25-hydroxyvitamin D; P = 0.40, 1,25-dihydroxyvitamin D). However, there was a significant inverse association among overweight and obese women for 25-hydroxyvitamin D levels (RR, 0.39; 95% CI, 0.16-0.93; P(trend) = 0.04). Further, those with adequate (>or=32 ng/mL) versus inadequate 25-hydroxyvitamin D levels had a modestly decreased risk of serous ovarian cancer (RR, 0.64; 95% CI, 0.39-1.05). Overall, our results do not suggest that plasma vitamin D levels are associated with risk of ovarian cancer. However, we observed significant associations in some subgroups, which should be evaluated further in other studies because increasing vitamin D intake is an easy preventive measure to adopt.