To evaluate pregnant women's knowledge about features of the second-trimester screening for Down syndrome and to assess if knowledge and educational level influence attitude towards amniocentesis prior to receiving test results. Three hundred pregnant Caucasian women younger than 35 years with no personal and/or family history of Down syndrome were surveyed. Women were randomized in two groups. One group of 150 women were surveyed by questionnaire prior to consultation with specially trained midwives, and another group of 150 women were surveyed after consultation. The questionnaire consisted of three sections: 1) demographic data of participants, 2) knowledge about prenatal screening for Down syndrome, 3) readiness to undergo amniocentesis for increased risk of Down syndrome. Women surveyed after consultation had greater total knowledge scores than those surveyed before consultation (p<0.001). A statistically significant difference in knowledge scores in relation to educational levels was observed only in women who were surveyed before consultation (p=0.007). Significantly more women were prepared to accept amniocentesis in the group surveyed after consultation (74%) than before consultation (53%) (p<0.001). Knowledge gained during prescreening consultation influenced pregnant women's attitudes towards further diagnostic investigation. A smaller proportion of women who were indecisive were obtained in the group surveyed after pre-screening consultation. Their indecisiveness was not affected by poor knowledge about screening, but rather by a difficulty to respond how they will feel and decide in the future if screening result will be positive.