Objectives We sought to evaluate pregnant women’s knowledge about features of second-trimester screening for Down syndrome and to assess whether knowledge and educational level influence their attitude toward amniocentesis before receiving test results. Methods Pregnant Caucasian women ( n = 300) <35 years old with no personal or family history of Down syndrome were surveyed. Women were randomized to 2 groups. One group of women ( n = 150) were surveyed by questionnaire before consultation with specially trained midwives; the other group of women ( n = 150) were surveyed after consultation. The questionnaire consisted of 3 sections: 1) participants’ demographic data, 2) knowledge about prenatal screening for Down syndrome, and 3) readiness to undergo amniocentesis if there was an increased risk of Down syndrome. Results Women surveyed after consultation had greater total knowledge scores than those surveyed before consultation ( p < .001). A statistically significant difference in knowledge scores in relation to educational levels was observed only in women who were surveyed before consultation ( p = .007). Significantly more women were prepared to accept amniocentesis in the group surveyed after consultation (74%) than before consultation (53%; p < .001). Conclusion Knowledge gained during a prescreening consultation influenced pregnant women’s attitudes toward further diagnostic investigation. A smaller proportion of women who were indecisive was observed in the group surveyed after prescreening consultation. Indecisiveness was not affected by poor knowledge about screening, but rather by difficulty in knowing how they will feel and what they will do if their screening result is positive.