The aim of this study was to determine whether there is increased leakage of neuron-specific enolase (NSE) and S-100 protein into amniotic fluid in pregnancies with neural tube defects, since both these proteins are produced by neural tissue, and to compare the value of these substances for detecting such defects with that of the more conventional techniques of alpha-fetoprotein (AFP) and acetylcholinesterase (AChE) gel electrophoresis. Amniotic samples from 25 mid-pregnancies (15-17 weeks' gestation) with neural tube defects (14 with open spina bifida and 11 with anencephaly) and from seven mid-pregnancies with abdominal wall defects were compared with a control material consisting of 80 amniotic fluid samples from 80 consecutive mid-pregnancy amniocenteses, with normal karyotypes and AFP concentrations. All of the above cases of abnormalities were primarily detected through increased AFP levels in the amniotic fluid. Amniotic fluid samples from 13 pregnancies with fetuses with autosomal chromosomal abnormalities and seven amniotic fluid samples contaminated with blood were also included in the investigation. It is concluded from the results that the conventional AFP assay combined with AChE gel electrophoresis is the best method for screening amniotic fluid for neural tube defects and defects of the abdominal wall. Neither NSE nor S-100 assay alone proved to be superior for the detection of these cases in mid-trimester amniotic fluid. The S-100 assay, however, could give additional information in cases where AChE gel electrophoresis is not decisive; for example, in samples contaminated with blood.