A rapid, direct radioimmunoassay for estriol-16alpha-(beta-D-glucuronide) in pregnancy urine has been developed by use of immunogens in which the estriol glucuronide hapten is linked through positions 2 or 4 of ring A to bovine serum albumin or agarose gel. The resultant antibodies cross react less than 1% with other estriol conjugates, estradiol conjugates, or unconjugated estrogens. Pregnanediol-3-glucuronide cross reacts less than 0.5%. This high degree of specificity of the antibodies allows for a 10,000-fold dilution of pregnancy urine to be assayed directly without interference from other steroids or urinary constituents. Good linear correlation was obtained for urinary-estriol glucuronide equivalents measured in the same subjects by our routine colorimetric method which separates and measures free estriol after hydrolysis. With an accelerated antibody-antigen equilibration of 20 minutes at 40 degrees, 10 samples can be easily processed in less than 90 minutes. The method also allows for the evaluation of variations in the excretion pattern of one estrogen conjugate in normal and high-risk pregnancies.