The concentration of unconjugated (‘free’) estriol (E3), estriol-3-sulfate, estriol-16-glucosiduronate (E316Gl) and estriol-3-sulfate-16-glucosiduronate (E3-SGl) in amniotic fluid was measured by fluorimetry using tritiated ‘free’ and conjugated E3 as internal standards. Determinations were carried out in uncomplicated pregnancies at elective repeat cesarean section and in severe Rh-isoimmunization prior to intrauterine transfusion at 29–32 weeks of gestation. In Rh-isoimmunization, a low total E3 concentration was found. This was accompanied by relatively reduced E3-16Gl and increased E3-SGl concentrations as compared to normal term pregnancy. The data do not permit to conclude that low amniotic fluid E3 levels are the result of impaired 16-glucosiduronation of E3.