A radioimmunoassay (RIA) for measuring total urinary estriol (E3) in pregnancy is described. Incubation of 0.1 ml urine for 30 minutes at 47°C with Helix pomatia enzyme containing both β glucuronidase and sulfatase activity, hydrolyzed estriol conjugates. Incubation of samples with E3-6,73H and diluted antiserum raised against estriol-6-one carboxymethyloxime coupled to bovine serum albumin, was carried out at 4°C for one hour. Recovery averaged 90 ± 6% (SD) for E3-16-glucuronide, 88.3 ± 5.7% for E3-3 glucuronide, and 84.6 ± 5.9% for E3-3 sulfate. The assay sensitivity was 25 pg, with a within and between assay coefficient of variation of 6.5% and 9.3% respectively. Results were unaffected by the presence of glucose, chlorothiazide, or hydrochlorothiazide. A close correlation was obtained between results by RIA and a specific colorimetric method. One technician can complete 50 samples within five hours.