Abstract: Objective: To assess folate and homocysteine status during normal pregnancy and postpartum in a longitudinal setting. Methods: This study, performed in 1995–1996, comprised 404 healthy pregnant Danish Caucasian women residential in Copenhagen County. Women taking folic acid tablets or vitamin B12 injections were not included. Dietary multivitamin supplements containing folic acid 100 μg or vitamin B12 1 μg, taken by 34%, were discontinued at inclusion. Participants had normal renal function. Folate status [erythrocyte (Ery-) folate, plasma (P-) folate, P-homocysteine] was measured at 18, 32 and 39 wk of gestation and 8 wk postpartum when the women were lactating. Results: Through 18, 32 and 39 wk of gestation and postpartum, P-folate demonstrated a significant fall: median values were 14.4, 10.2, 9.3 and 8.9 nmol/L, respectively (P < 0.0001). The prevalence of low P-folate <6 nmol/L increased during pregnancy from 0.7% to 19.0% postpartum (P < 0.0001). Ery-folate displayed a similar, significant fall: median value was 0.84, 0.75, 0.65 and 0.55 μmol/L, respectively (P < 0.0001). The prevalence of low Ery-folate <0.40 μmol/L increased during pregnancy from 0.5% to 17.2% postpartum (P < 0.0001). P-homocysteine demonstrated a significant increase: median value was 6.4, 7.0, 7.7 and 10.8 μmol/L, respectively (P < 0.0001). The prevalence of P-homocysteine >13 μmol/L increased during pregnancy from 0.7% to 20.8% postpartum (P < 0.0001). The prevalence of low folate status (defined as P-folate <6 nmol/L and P-homocysteine >13 μmol/L) was 0%, 0%, 1.2%, and 8.4% at 18, 32 and 39 wk of gestation and 8 wk postpartum, respectively. Conclusion: Low folate status occurs among Danish pregnant women, especially in late pregnancy and postpartum during lactation. Despite new guidelines for folic acid supplement since 1997, only 13% of pregnant women followed the guidelines in 2003. The official recommendations for periconceptional folic acid supplement should be reconsidered and reinforced.