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The detection of fatty acid ethyl esters (FAEE) in meconium may provide an objective estimate of prenatal alcohol exposure independent of maternal history. The authors report the results of the first population-based study conducted to investigate basal FAEE levels in the meconium of neonates not exposed to alcohol. Two hundred seven nondrinking women and their neonates were recruited from Toronto and Jerusalem. FAEE were extracted from meconium by solid-phase extraction and analyzed by GC/FID. Similar procedures were conducted in six neonates born to confirmed heavy drinkers. Low levels of meconium FAEE were detected from both cohorts (mean, 1.37 nmol/g vs. 2.08 nmol/g, Toronto vs. Jerusalem). Ethyl stearate, oleate, and linoleate were below the limit of detection in >80% of all samples, whereas ethyl laurate and palmitate were detected in >50% of the samples. Ethyl myristate was the FAEE most commonly detected (>80%). All six meconium samples with confirmed maternal drinking histories tested positive for FAEE at significantly higher levels (mean, 11.08 nmol/g). The use of 2 nmol total FAEE/g meconium as the positive cutoff, when lauric and myristic acid ethyl esters were excluded, yielded the greatest sensitivity (100%) and specificity (98.4%). The authors conclude that certain FAEE are present at measurable levels in the meconium of neonates not exposed to maternal drinking, and correction is needed to allow high specificity.