Objective: It was the aim of this study to investigate the pregnancy characteristics that influence the measured concentrations of maternal serum-free β-human chorionic gonadotropin (β-hCG) and pregnancy-associated plasma protein-A (PAPP-A) at 9+0–13+6 weeks’ gestation. Methods: In singleton pregnancies attending for routine care, serum-free β-hCG and PAPP-A were measured at 9+0–13+6 weeks’ gestation and fetal nuchal translucency was measured at 11+0–13+6 weeks. The population included 27,908 chromosomally normal and 104 trisomy 21 pregnancies. Multiple regression analysis was used to examine the pregnancy characteristics that have a significant effect on the measured concentrations of free β-hCG and PAPP-A. We also examined the impact of incorporating temporal effects on performance of screening for trisomy 21. Results: Serum-free β-hCG and PAPP-A concentrations were significantly affected by gestational age, maternal weight, racial origin, parity, preexisting diabetes mellitus type 2, smoking and conception by in vitro fertilization. There was a significant gestational age-dependent effect of Afro-Caribbean race on PAPP-A levels (p = 0.0005), with a weekly increase of 4.9% (95% CI 2.1–7.8). Conclusions: Serum-free β-hCG and PAPP-A concentrations at 9+0–13+6 weeks’ gestation are affected by several pregnancy characteristics and the effect of Afro-Caribbean race on PAPP-A increases with gestational age.