Objective: To determine the content of decision-relevant knowledge needed for informed decision-making about (non-) participation in prenatal screening for Down's syndrome (DS), in order to develop a knowledge questionnaire for routine application in large-scale programme evaluations. Methods: A generic list of content domains for knowledge about screening was extracted from the literature. Items reflecting specific knowledge domains were constructed. An expert group of professionals and pregnant women expressed whether domains and items represented decision-relevant information. Results: All presented domains were scored as (very) important. Options when receiving an 'increased probability for DS' test result, the meaning of this result, the aim of the screening, and voluntary nature of the test were scored as most important. The condition being screened for, prevalence, and the screening procedure were scored as relatively less important, with a high amount of expert consensus. Conclusion: A knowledge measure for prenatal screening for DS was developed, based on domains and items acquired by expert consensus. Practice implications: This measure of decision-relevant knowledge can be used in routine, large-scale evaluations of the procedure for offering information about prenatal screening for DS. (C) 2010 Elsevier Ireland Ltd. All rights reserved.