A reliable method for the estimation of urinary estriol in pregnancy is presented. This method is based upon acid hydrolysis, solvent partition, methylation and chromatography. Estriol is measured by colorimetry using the Kober reaction. All results are corrected for procedural loss by adding tritium-labelled estriol-16-glucosiduronate to the unhydrolyzed urine. This method proves to be sufficiently specific, accurate and precise. One operator may complete six assays within one working day. It is emphasized that a comprehensive recovery control which includes the initial hydrolytic procedure is essential if estriol determinations are to serve as a diagnostic aid in obstetrics.