The most reliable tool at present for the prenatal detection of open neural tube defects is the amniotic fluid alpha-fetoprotein assay. Nevertheless its nonspecificity and false-positive results produced by fetal blood admixture and other less well-defined causes have spurred the search for a more specific test. Increased amniotic fluid acetylcholinesterase activity has been reported in association with open neural tube defects. The authors have assayed acetylcholinesterase iri second-trimester amniotic fluid samples with previously determined alpha-fetoprotein values from 200 pregnancies with 96.5% of the outcomes known. These pregnancies were selected because of known fetal abnormalities or difficulties in prenatal diagnosis. High alpha-fetoprotein and acetylcholinesterase activity was detected in all 66 cases of open neural tube defects. The authors were able to reclassify correctly 89% of normal pregnancies with spuriously high alpha-fetoprotein by demonstrating no acetylcholinesterase band after slab gel electrophoresis. Nine cases with a normal outcome but high alpha-fetoprotein values and acetylcholinesterase activity constitute the remaining false-positive results. Notwithstanding the false-positive results, which may have been due to undetected fetal blood admixture, the acetylcholinesterase gel electrophoresis assay is clearly a valuable adjunctive test to the alpha-fetoprotein assay for the prenatal detection of neural tube defects. Although not specific for open neural tube defects, acetylcholinesterase analysis by gel electrophoresis of amniotic fluid is significantly less influenced by fetal blood than is alpha-fetoprotein and, furthermore, may prove as reliable a diagnostic test for open neural tube defect.