A high-performance liquid chromatography (HPLC) method with mass spectrometric detection is described for determination of arecoline in newborn meconium, urine and cord serum, using pilocarpine as internal standard. The analytes were extracted from neonatal biological matrices with chloroform/isopropanol (95:5, v/v) at alkaline pH. Extracts were analyzed by HPLC coupled to an electrospray (ESI) interface and a quadrupole mass spectrometer. Chromatography was performed on a C8 reversed-phase column using 10 mM ammonium acetate (pH 4.3)/acetonitrile (90:10, v/v) as mobile phase. The mass spectrometer was operated in selected ion monitoring mode. The method was validated over the concentration range 0.005-1.00 μg/g meconium, 0.004-1.00 μg/mL cord serum and 0.001-1.00 μg/mL urine. Mean recoveries ranged between 86.5 and 90.7% for arecoline in the different biological matrices, with precision always better than 10%. The quantification limits of arecoline were 0.005 μg/g meconium, 0.004 μg/mL cord serum, and 0.001 μg/mL urine. The method was applied to the analysis of neonatal biological matrices to assess eventual fetal exposition to arecoline. Two newborns from Asian mothers who declared areca nut consumption presented arecoline in meconium with concentrations in the range 0.006-0.008 μg/g; also the urine from one neonate tested positive for the drug. Copyright © 2003 John Wiley & Sons, Ltd.