Background: Folate and choline are essential nutrients of particular importance during pregnancy and lactation and among nonpregnant women of reproductive age. However, in the era of folic acid (FA) fortification, high folate status and supplement use during pregnancy have been linked to adverse outcomes including asthma and eczema in children. No dose response studies conducted with women informed the choline Adequate Intakes (AIs), meanwhile evidence suggests that intakes greater than the current AI could optimize pregnancy endpoints. Research informing intake recommendations and strategies that promote optimal outcomes while avoiding inadequate and excessive intakes of folate and choline are needed. Objective: This research sought to quantify status and functional indicators of folate and choline metabolism in response to controlled intakes among pregnant, lactating, and nonpregnant women. Design: Pregnant (n=26), lactating (n=28), and nonpregnant (n=21) women consumed: a prenatal supplement containing 750 [MICRO SIGN]g FA; 480 or 930 mg choline/d ; and 200 mg docosahexaenoic acid (DHA)/d for 10-12 wks. Biological samples were collected throughout the study. Folate status indicators were measured in all groups. Phosphatidylcholine(PC)-DHA, a functional indicator of choline metabolism, was assessed in pregnant and nonpregnant women. Results: (1) At study-end, serum folate concentrations did not differ (P=0.876) by physiologic group and urinary folate excretion represented ~9-43% of intake. Breastmilk FA concentrations increased (P=0.003), while total folate remained constant (P= 0.244) over time. (2) PC-DHA was greater (P=0.01-0.001) among pregnant women at baseline. PCDHA increased (P<0.001) in both groups regardless of choline intake; however, among nonpregnant women, consumption of 930 mg choline/d led to greater (P=0.011) PC-DHA. Conclusions: (1) The study folate dose yielded supranutritional folate status in all physiologic groups. Given unresolved concerns about exposure to excess FA and the widespread folate adequacy of our FA-fortified population, it may be prudent to reduce the amount of FA in prenatal supplements. (2) Via its capacity to donate methyl groups, a higher choline intake may facilitate synthesis and delivery of DHA to peripheral tissues among nonpregnant women and increase availability of methyl groups during pregnancy. These beneficial endpoints of choline intakes greater than the current AIs warrant further exploration.