Evidence suggests that nutrients involved in one-carbon metabolism are implicated in ovarian cancer etiology. No studies have evaluated the role of choline, and that of its metabolite, betaine. We prospectively examined the relationship between the intake of these nutrients and ovarian cancer risk among 159 957 participants from the Nurses' Health Study (NHS) and NHSII. The average nutrient intake was assessed every 2-4 years beginning in 1984 (for NHS) and in 1991 (for NHSII). With up to 22 years of follow-up per cohort, 526 incident cases of ovarian cancer were diagnosed. There were no associations between total choline, betaine, as well as choline plus betaine intake and ovarian cancer risk (for example, relative risk, top vs bottom quintile of choline=0.98; 95% confidence interval: 0.73-1.31; P(trend)=0.81). Results did not vary by alcohol consumption, folate intake or after the exclusion of cases diagnosed during the 4-year period after dietary assessment. These data provide little evidence for a role of these nutrients in ovarian cancer etiology.