Methods for the determination of five steroids in amniotic fluid are described. The following steroids have been measured in amniotic fluid of 29 normal pregnancies: dehydroepiandrosterone, 11.1 μg/l; pregnanediol, 164.4 μg/l; 16-ketoandrostenediol, 214.7 μg/l; 16α-hydroxydehydroepiandrosterone, 599.6 μg/ and oestriol, 674.3 μg/l. Analyses in eight pregnancies complicated by diabetes, polyhydramnios, anencephaly and severe Rh-isoimmunization reveal low or nondetectable amounts of oestriol, 16-ketoandrostenediol and 16α-hydroxydehydroepiandrosterone, The concentrations of dehydroepiandrosterone and pregnanediol appear not to be significantly influenced under these circumstances unless intrauterine foetal death has already occurred. This study seems to offer an explanation for the differences of oestriol concentrations in amniotic fluid, maternal plasma and urine in pregnancies complicated by severe Rh-isoimmunization.