BACKGROUND Folate dose-response studies in women of childbearing age who consumed a folic acid (FA)-containing multivitamin in the era of FA fortification are lacking. OBJECTIVE We sought to investigate folate-status response to a known folate dose comprising an FA-containing prenatal supplement (750 μg/d) plus natural food folate (400 μg/d) in third-trimester pregnant women, lactating women 5-15 wk postpartum, and nonpregnant women. METHODS Pregnant (n = 26), lactating (n = 28), and nonpregnant (n = 21) women consumed the study folate dose under controlled intake conditions for 10-12 wk. Blood, urine, and breast milk were collected at baseline, study midpoint, and study end. RESULTS Study-end serum total folate concentrations averaged ~30 ng/mL and did not differ by physiologic group (P = 0.876). Study-end urinary folate excretion represented ~9-43% of total folate intake and ranged from 100 to 500 μg/d. Third-trimester pregnant women excreted less urinary folate than did lactating (P = 0.075) and nonpregnant (P < 0.001) women. Lactating women excreted less (P < 0.001) urinary FA than did nonpregnant women. Breast-milk total folate concentrations remained constant (P = 0.244; 61.8 ng/mL at study end), whereas breast-milk FA concentrations increased (P = 0.003) to 24.1 ng/mL at study end. CONCLUSIONS The consumption of the study folate dose yielded a supranutritional folate status regardless of the physiologic state. Based on urinary folate excretion, folate use was greatest to least: pregnant > lactating > nonpregnant women. Breast-milk folate species were responsive to maternal folate intake, and FA made up ~40% of breast-milk total folate at study end. These findings warrant revisiting prenatal supplement FA formulation in populations exposed to FA-fortification programs.