This prospective study investigates the relationship between insulin-dependent diabetes and maternal serum levels of alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and human chorionic gonadotropin (hCG). It also examines the potential impact on screening for Down syndrome. The population-based cohort included 20,321 pregnant women in Maine who underwent routine serum screening for Down syndrome in the second trimester. The cohort included 52 women with insulin-dependent diabetes. Maternal serum AFP levels are now routinely adjusted for insulin-dependent diabetes. These adjustments, therefore, were made routinely in the diabetic women, but no equivalent adjustments were made for uE3 and hCG values. The initial false-positive rate (using all three markers) among the women with diabetes was not significantly different from that in the non-diabetic population (7.7 and 5.4 per cent, respectively). Prior to adjustment for insulin-dependent diabetes, the median AFP level in the 52 women was 0.73 multiples of the median (MOM); the median levels of uE3 and hCG were 0.93 and 0.98 MOM, respectively. When the uE3 and hCG levels were adjusted, the initial false-positive rate was unchanged. Median serum levels of uE3 were significantly higher in the 33 women whose onset of diabetes was prior to 19 years of age (0.99 MOM) than in the 19 women whose onset of diabetes was at age 19 or older (0.84 MOM). This is the first population-based study to investigate the relationship between diabetes and serum levels of AFP, uE3, and hCG, and confirms earlier observations from a case-control study that found only slightly lower uE3 and hCG levels.