Abstract Background: In many countries, current recommendations are that women take a daily 400-μg folic acid supplement from before conception until the end of the 12th week of gestation for the prevention of neural tube defects. Low folate status is associated with an increased concentration of plasma total homocysteine (tHcy), a risk factor associated with pregnancy complications such as preeclampsia. Methods: In a longitudinal study, we determined tHcy and corresponding folate status in 101 pregnant women at 12, 20, and 35 weeks of gestation, in 35 nonpregnant controls sampled concurrently, and in a subgroup (n = 21 pregnant women and 19 nonpregnant controls) at 3 days postpartum. Results: Plasma tHcy was significantly lower throughout pregnancy compared with nonpregnant controls, with values lowest in the second trimester before increasing toward nonpregnant values in the third trimester. Importantly, mean tHcy concentrations were lower in pregnant women taking folic acid supplements than in those not, an effect that reached significance in the third trimester (5.45 vs 7.40 μmol/L; P <0.05). During the third trimester, tHcy concentrations were significantly higher in pregnant women with a history of miscarriage than in women with no previous history (8.15 vs 6.38 μmol/L; P <0.01). Conclusions: This is the first longitudinal study to show that homocysteine concentrations increase in late pregnancy toward nonpregnant values; an increase that can be limited by enhancing folate status through continued folic acid supplementation. These results indicate a potential role for continued folic acid supplementation in reducing pregnancy complications associated with hyperhomocysteinemia.