We describe a radioimmunoassay for total urinary estriol in pregnancy. A 25-mul aliquot of the urine specimen is acid hydrolyzed, neutralized, and diluted before assay. We use rabbit antisera against estriol-6-(O-carboxymethyl)oxime/bovine serum albumin and ammonium sulfate precipitation at room temperature. Results are unaffected by glucose or methenamine mandelate, a urinary tract antiseptic. Using semi-automatic pipetting equipment, one laboratory technologist can complete 50 assays within 8 h. This technique is both reliable and convenient and should decrease the expense of routine estriol assays.