OBJECTIVE: Our goal was to prospectively evaluate the use of the free β-subunit of human chorionic gonadotropin and dimeric inhibin A for the detection of fetal Down syndrome and other aneuploidies. STUDY DESIGN: Women who had a second-trimester multiple-marker screening test (α-fetoprotein, unconjugated estriol, human chorionic gonadotropin) and genetic amniocentesis from August 1996 to August 1998 were included. Serum was also analyzed for inhibin and the free β-subunit of human chorionic gonadotropin. Detection and false-positive rates for 4 analyte combinations at 5 different screening risk cutoff points for Down syndrome were determined and compared. RESULTS: We evaluated 1256 patients, including 23 with aneuploidy (13 with Down syndrome, 10 others). The maternal age was 35.9 ±4.6 years (mean ± SD). At the optimal risk cutoff point for Down syndrome detection (1:190; false- positive rate, 19%), the multiple-marker screening test plus inhibin was superior, detecting 85% of Down syndrome cases, in comparison with 69% when the multiple-marker screening test alone was used and 62% when the other 2 combinations were used. The multiple-marker screening test plus inhibin also detected 60% of the other aneuploidies. CONCLUSIONS: When evaluated prospectively in a high-risk population, the multiple-marker screening test plus inhibin was superior to the traditional multiple-marker screening test and 2 other analyte combinations, with a lower false-positive rate and increased detection of all aneuploidies in a high-risk population.