OBJECTIVES: The primary aim of this study was to assess pregnant women's knowledge of first trimester combined Down's syndrome screening in a setting of required informed consent. Secondary, we wanted to identify relevant differences in knowledge level among subgroups of pregnant women, including subgroups informed in different ways about prenatal examinations. METHODS: Data stem from a population-based cross-sectional questionnaire study including 15 multiple-choice questions assessing knowledge of different screening aspects. Included were 6,427 first trimester pregnant women from three Danish obstetric departments offering prenatal screening free of charge. Both participants and non-participants in the screening programme were included. The results are based on 4,095 responders (64%). Differences between sub-groups were examined by chi-squared tests and logistic regression analysis. Estimates are stated with 95% CI. RESULTS: The majority of the participants (87.6% (86.6-88.6) to 92.6% (91.7-93.3)) correctly identified the test concept and the main condition being screened for. Fewer participants (16.4% (15.3-17.6) to 43.3% (41.8-44.8)) correctly recognised test accuracy and the potential risk of adverse findings other than Down's syndrome. Knowledge level was positively associated with length of education (adjusted ORs 1.0 (0.8-1.4) to 3.9 (2.4-6.4)) and participation in the screening programme (adjusted OR 0.9 (0.6-1.3) to 5.9 (3.9-8.8)). Participation in an individual information session was weakly associated with more knowledge. CONCLUSION: The majority of the pregnant women correctly identified the test concept and the main condition being screened for. The pregnant women were found less knowledgeable on test accuracy and drawbacks. Copyright © 2010 ISUOG. Published by John Wiley & Sons, Ltd.