We aimed to compare the levels of alpha-fetoprotein (AFP) and free b-human chorionic gonadotrophin (b-hCG) levels as multiples of the median (MoM) values between spontaneous and in vitro fertilized (IVF) twin pregnancies. The control group of spontaneous singleton pregnancies was used for calculating the gestational age speci®c median levels of the values. Within a cohort of 19 310 pregnancies, 145 twin pregnancies were identi®ed. The data were collected from Down syndrome (DS) screening programmes in four University catchment areas in Finland between 1994±98. Maternal midtrimester serum marker levels were measured across gestational weeks 14±18. There were no fetal chromosome anomalies in either of the twin groups or the singleton group. Serum AFP of 145 and b-hCG values of 39 spontaneous twin pregnancies were compared to the values of 6548 singleton pregnancies. In IVF twins 30 AFP and 29 b-hCG values were compared to the levels of the control group. Both AFP and b-hCG values were twice as high in the spontaneous twin pregnancies (medians 2.18 and 1.83 MoM respectively) as in the singleton group (medians 1.00 and 1.00 MoM respectively). In IVF twin pregnancies b-hCG levels were higher (median 2.20 MoM) than in spontaneous twins ( p=0.08), whereas no signi®cant difference was found in AFP levels (2.30 MoM). In conclusion, the higher levels of b-hCG levels in IVF twin pregnancies should be considered in DS screening to avoid high false positive rates.