Folic acid deficiency during pregnancy can lead to neural tube defects (NTD) in the fetus. Folate status was determined in a representative sample of Belgian pregnant women and determinants of folate status were assessed.The women were selected using a multi-stage proportionate-to-size sampling design. Blood samples were collected and a questionnaire was completed face-to-face with a study nurse. Erythrocyte (red blood cell (RBC)) folate concentration was measured by chemoluminescence.In total, 1311 pregnant women participated and women with a lower socio-economic status were well represented. Median RBC folate concentration was 436 ng/ml (95% confidence interval=425-452 ng/ml) among first trimester and 496 ng/ml (95% confidence interval=474-515 ng/ml) among third trimester women. Few women had a RBC folate concentration below 140 ng/ml, indicating depletion of folate stores. In the first trimester, 39% of women had a RBC concentration below 400 ng/ml, whereas 15% of the first trimester women had a RBC concentration below 300 ng/ml. Among women in the first trimester, 69.1% reported taking folic acid-containing supplements of which 41.2% started taking them before pregnancy. For third trimester women, these percentages were 76.2% and 21.9%, respectively. In both trimesters, folate status increased significantly with education level and was significantly higher among women who planned the pregnancy and who did not smoke.It was found that 39% of the first trimester pregnant women had a folate status that might not be optimal to prevent NTD. Some groups of women need to be targeted as they are at higher risk of inadequate folate status.