Our objective was to measure amniotic fluid amino acid concentrations in pregnant women diagnosed as having fetuses with spina bifida in the second trimester of pregnancy. Fifteen pregnant women who had fetuses with spina bifida detected by ultrasonography (spina bifida group) in the second trimester and 19 women who had abnormal triple screenings indicating an increased risk for Down’s syndrome but had healthy fetuses (control group) were enrolled in the study. Amniotic fluid was obtained by amniocentesis. The chromosomal analysis of the study and control groups was normal. Levels of free amino acids were measured in amniotic fluid samples using EZ: fast kits (EZ: fast GC/FID free (physiological) amino acid kit) by gas chromatography (Focus GC AI 3000 Thermo Finnigan analyzer). The mean levels of alanine, cystathionine, cysteine, phenylalanine, tryptophane, and tyrosine amino acids were found to be significantly higher in fetuses of the control group than in the spina bifida group (p < 0.05). The detection of significantly higher amino acid concentrations in the amniotic fluid of healthy fetuses suggests loss of amino acids from the fetus through the spinal cord may contribute to the etiology of spina bifida.