A method is described for the assay of estriol conjugates in 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 the fluid. After removal of proteins, the conjugates are separated on a Celite partition column. Each conjugate is hydrolyzed with Glusulase and the E3 purified by chromatography on Sephadex LH-20. Assay is by fluorescence. The method was applied to 7 samples obtained from midpregnancy and 7 samples from term pregnancy. The total E3 was 0.84 to 9.3 μg/ml in the former and 74 to 311 μg/100 ml in the latter. The most important relative difference was in the E3-16G to E3-3S-16G ratios. They were greater than 2 at term but only about 0.5 at midpregnancy. No significant changes in the percentage contribution of E3-3S and E3-3G were noted as gestation progressed.