Objective: To assess the ability of second-trimester maternal serum free β-hCG to detect fetal Down syndrome and to compare free β-hCG to intact hCG in the multiple-marker screening test for Down syndrome. Methods: From our bank of stored maternal sera, we selected 40-50 samples from euploid pregnancies at each week of gestation from 14 to 20 weeks and 31 samples from Down syndrome pregnancies. Free β-hCG was measured by enzyme-linked immunosorbent assay, and week-specific multiples of the median (MoM) were derived. The free β-hCG Down syndrome detection and false-positive rates were determined. Free β-hCG was then substituted for intact hCG in the multiple-marker screening test, and the Down syndrome detection and false- positive rates at various risk cutoffs were compared. Results: The mean (± standard deviation) maternal age of all study samples was 35.6 ± 5.3 years. The mean Down syndrome free β-hCG MoM was significantly higher than the mean euploid MoM (2.4 ± 1.1 versus 1.2 ± 1.0; P < .001). A free β-hCG level of at least 1.7 MoM identified 68% of Down syndrome pregnancies at a false- positive rate of 20%. When intact hCG was replaced wit free β-hCG in the multiple-marker screening test, a higher Down syndrome detection rate was achieved at a lower false-positive rate at each of several screen positive risk cutoffs. Conclusion: Elevated free β-hCG levels identify Down syndrome pregnancies. Replacing intact hCG with free β-hCG in the multiple-marker screening test results in a higher Down syndrome detection rate at a lower false-positive rate.