Prenatal diagnosis of esophageal atresia (EA) may improve the outcome of affected neonates by allowing optimization of both prenatal and postnatal care. Prenatal sonographic detection is based on polyhydramnios and/or nonvisualization of the fetal stomach bubble, two signs with a large number of etiologies. We evaluated a biochemical approach to improving diagnostic efficiency. We compared amniotic fluid biochemical markers in 44 EA cases with 88 polyhydramnios and 88 nonpolyhydramnios controls. Both matched for GA with cases. Total proteins, alpha-fetoprotein (AFP), and digestive enzyme activities were assayed, including gamma-glutamyl transpeptidase (GGTP). We defined an EA index (AFP multiplied by GGTP). A significant difference (p Ͻ 0.0001) was observed for total protein, AFP, GGTP, and EA index between the EA group and each of the two control groups. No statistical difference was observed for any marker between the two most frequent EA subgroups (type I and type III) or between the two control groups. Using a cutoff of 3 for the EA index, 98% sensitivity and 100% specificity were observed for amniotic fluid prenatal diagnosis of EA, whatever the anatomical type. A large prospective series is required to confirm these results.