Objective To establish an algorithm for first-trimester combined screening for trisomy 21 with biochemical testing from 7 to 14 weeks' gestation and ultrasound testing at 11-13 weeks. Methods This was a multicenter study of 886 pregnancies with trisomy 21 and 222 475 unaffected pregnancies with measurements of free β-human chorionic gonadotropin (β-hCG) and pregnancy-associated plasma protein-A (PAPP-A) at 7-14 weeks' gestation. Multiple regression modeling of log-transformed marker values was used to produce log multiples of the median (MoM) values for PAPP-A and free β -hCG. The models included terms for the center attended and the machine used for biochemical analysis, gestational age, maternal racial origin, maternal weight, smoking status and method of conception. Bivariate Gaussian distributions were fitted to log MoM PAPP-A and log MoM free β3-hCG in trisomy 21 and in unaffected pregnancies. In each case the patient-specific risk for trisomy 21 was estimated by multiplying the individual maternal age-related risk with the likelihood ratio (LR) for fetal nuchal translucency (NT) according to the mixture model and the combined LR for maternal serum free β-hCG and PAPP-A. Estimates of detection rates for trisomy 21 and false-positive rates were calculated for combined screening with measurements of NT at 12 weeks together with measurements of free β-hCG and PAPP-A from 8 to 13 weeks. Results In trisomy 21 pregnancies the mean log MoM free β-hCG increased linearly with gestation between 7 and 14 weeks, whereas the relation between log MoM PAPP-A and gestation was fitted by a quadratic equation such that the maximum separation between trisomy 21 and unaffected pregnancies occurs at 9-10 weeks. At a false-positive rate of 3% the detection rate of combined screening at 12 weeks was 86% and this increased to 90% by biochemical testing at 9 weeks and ultrasound scanning at 12 weeks. The detection rate increased to 92% by measuring PAPP-A at 9 weeks and free β-hCG at the time of the scan at 12 weeks. Conclusion The performance of first-trimester biochemical screening for trisomy 21 is best at 9-10 weeks rather than at 7-8 or 11-14 weeks.