To investigate maternal serum unconjugated oestriol (uE3) and human chorionic gonadotrophin (hCG) levels in twin pregnancies and to consider the implications of the results for antenatal screening for Down's syndrome. Measurement of maternal serum uE3 and hCG levels from 15-22 weeks of gestation in twin and singleton pregnancies. Previously available maternal serum alpha-fetoprotein (AFP) levels were also presented. Stored serum samples collected from women receiving routine antenatal care in Oxford were used. 200 women with a twin pregnancy and, for each, three singleton control pregnancies matched for gestational age (same completed week of pregnancy) and duration of storage of the serum sample (same calendar quarter). The median uE3, hCG and AFP levels in the twin pregnancies were respectively, 1.67 (95% CI 1.56-1.79), 1.84 (95% CI 1.64-2.07) and 2.13 (95% CI 1.97-2.31) multiples of the median (MoM) for singleton pregnancies at the same gestational age. The variance of values for the three serum markers (expressed in logarithms), and the correlation coefficients between any two, were similar in the twin and singleton pregnancies. In maternal serum screening programmes for Down's syndrome dividing uE3, hCG and AFP MoM values in twin pregnancies by the corresponding medians for twin pregnancies will, in expectation, yield a similar false-positive rate in twin pregnancies as in singleton pregnancies.