Background.Studies have shown that folic acid supplementation in early pregnancy markedly reduces the risk of neural tube defects (NTDs). Investigation of the relation between relative dose of supplemental folic acid or total folate intake and NTD risk is limited.Methods.We used data from 23,228 women, predominantly from the northeastern United States, enrolled between October 1984 and June 1987 in a prospective study of early prenatal exposures and pregnancy outcomes. Diet and vitamin intake data were gathered in the early second trimester. NTDs were ascertained through prenatal testing and by report of the delivering physician. Data analyses included multiple logistic regression and restricted spline regression modeling.Results.For each additional 500 dietary folate equivalents consumed per day, the prevalence of NTDs decreased by 0.78 cases (95% confidence interval [CI] = 0.47–1.09) per 1,000 pregnancies. Compared with women having the lowest total folate intakes (0–149 folate equivalents per day), the prevalence of NTDs declined by 34%, 30%, 56% and 77% among the offspring of those women consuming 150–399, 400–799, 800–1199 and ≥ 1200 folate equivalents per day, respectively (P-value for linear trend = 0.016).Conclusions.Our results suggest that NTD risk declines markedly with modest increases of total folate in early pregnancy. Total folate dose, rather than supplemental folate alone, should be considered in formulating public health guidelines for NTD prevention.