OBJECTIVES: To study associations between pregnant women's knowledge of prenatal screening and decisional conflict in deciding whether to participate in first trimester screening for Down's syndrome in a setting of required informed consent and to study associations between knowledge and personal well-being respectively worries in pregnancy. METHODS: A population-based cross-sectional study with 6,427 pregnant women consecutively included before the time of a nuchal translucency scan. Participants were recruited from three Danish obstetric departments offering prenatal screening free of charge. The results presented are based on 4,111 pregnant women (64%). Knowledge was measured by 15 knowledge questions. The primary outcomes were measured by use of pre-existing validated scales i.e. The Decisional Conflict Scale, the WHO well-being index, and the Cambridge Worry Scale. Associations were analysed by multivariate logistic and linear regression analyses. RESULTS: A higher level of knowledge was associated with less decisional conflict when deciding whether to participate in first trimester Down's syndrome screening (adjusted odds ratio 1.31 (1.26 to 1.37)). An increased level of knowledge was also associated with higher levels of well-being (adjusted linear coefficient 0.51 (0.26 to 0.75), p < 0.001). Knowledge was not associated with worries, neither in general nor specifically about something being wrong with the baby. CONCLUSION: The results of this study indicate the importance of ensuring a high level of knowledge for the pregnant women making choices about participation in prenatal screening for Down's syndrome in order to improve psychological management of test decisions. Copyright © 2010 ISUOG. Published by John Wiley & Sons, Ltd.