OBJECTIVE: The purpose of this study was to determine, among six second- trimester maternal serum analytes, the best three-analyte combination for fetal Down syndrome detection. STUDY DESIGN: With use of commercially available assay kits medians for free β-human chorionic gonadotropin, CA 125, and dimeric inhibin A were established in sterno sera from 45 to 50 euploid pregnancies at each week of gestation from 14 to 22 weeks and from 33 Down syndrome pregnancies. Maternal serum α-fetoprotein, unconjugated estriol, and intact human chorionic gonadotropin levels measured in each sample before storage were retrieved. All 20 possible three-analyte combinations were evaluated in the multiple-marker screening test for Down syndrome. RESULTS: The mean maternal age of the study population was 35.6 ± 5.3 years. The best three-analyte combination was maternal serum α- fetoprotein, free β-human chorionic gonadotropin, and dimeric inhibin A: 97% of Down syndrome cases were detected at a false-positive rate of 16%. At a slightly higher false-positive rate (18%) maternal serum α-fetoprotein, estriol, and intact human chorionic gonadotropin detected only 79% of cases. CONCLUSIONS: Of six second-trimester maternal serum analytes, the best three- analyte combination for fetal Down syndrome detection was maternal serum α- fetoprotein, free β-human chorionic gonadotropin, and dimeric inhibin A. This retrospective analysis should now be confirmed prospectively.