In this relatively simple, rapid assay of estriol-16 alpha-glucuronide in pregnancy urine, the urine sample is diluted 20-fold with phosphate buffer (pH 5.2) containing 360 mL of acetonitrile and 2 g of cetyltrimethylammonium bromide per liter, then directly injected into the chromatograph. A sample can be assayed within 14 min. Day-to-day CVs ranged from 2.3% at 45 mg/L to 2.9% at 4.8 mg/L. The limit of sensitivity is 0.4 mg/L. Results by the present method (y) correlated and compared very well with those by a method involving fractionation of estrogen conjugates and gas chromatography (x) for 24 samples of pregnancy urine (y = 1.09x + 0.303; r = 0.947). This assay is inexpensive and suitable for complete automation.