A method is described for the assay of estriol (E3) conjugates in human amniotic fluid. Tritiated estriol-3-sulfate (E3-3S), estriol-16-glucosiduronate (E3-16G), estriol-3-glucosiduronate (E3-3G), and estriol-3 sulfate-16-glucosiduronate (E3-3S-16G) are added to amniotic fluid. After precipitation of the proteins, the conjugates are converted to their triethylammonium salts and separated on a Sephadex LH-20 column. Following enzyme hydrolysis with Glusulase, the estriol is submitted to radioimmunoassay using an ovine anti-E3 serum, which is covalently linked to 1–4 μ diameter glass particles. The method was applied to amniotic fluid from normal and Rh-isoimmunized patients from 28–40 weeks of gestation. In both series, sequential samples were obtained from 5 individual patients as well as single samples from other patients. Each normal patient followed sequentially exhibited a progressive rise in the E3-16G/E3-3S-16G ratio. This was not observed in the Rh-isoimmunized patients followed serially. Thesignificance of this ratio in terms of fetal well-being and maturation has yet to be elucidated.