Background. The hormonal milieu associated with pregnancy has become a focus of interest owing to potential links with the developmental origins of health and disease. Phytoestrogens are hormonally active plant-derived chemicals that may have an impact on human reproductive processes. However, developmental exposure to phytoestrogens has not been well characterized and thus our objective was to quantify phytoestrogen exposure during pregnancy and lactation. Methods. Women in the second trimester of pregnancy entered the study during counseling for prenatal genetic information. Women who had an indication for a genetic amniocentesis on the basis of late maternal age were approached for inclusion. They completed an environmental questionnaire; a sample of amniotic fluid was collected for karyotype, blood was collected from women during pregnancy and at birth, from the umbilical cord and breast milk. Samples were tested for the presence of daidzein and genistein by GC Mass Spectroscopy. Findings. Phytoestrogens are commonly found in pregnant women’s serum and amniotic fluid during pregnancy. There is a sex difference in the concentrations with higher levels in amniotic fluid containing female fetuses. This difference was not present in maternal serum. Soy ingestion increases amniotic fluid phytoestrogen concentrations in female and male fetuses. The presence and concentrations of phytoestrogens did not differ in relation to common pregnancy complications or preexisting infertility.