Catechol estrogens such as 2-OH estrone are interesting estrogen metabolites formed in several human tissues and excreted in urine. We developed and thoroughly validated a radioimmunoassay for urinary 2-OH estrone that has several advantages over published RIAs. Because we developed a relatively specific antiserum, we did not include a preliminary chromatographic step to eliminate cross-reacting urinary steroids. We hydrolyzed urinary steroid conjugates with beta-glucuronidase from Helix pomatia because recoveries after acid hydrolysis were only 49.6% compared with 73.8% after enzyme hydrolysis. Published RIAs for urinary 2-OH estrone use acid hydrolysis. Our RIA measured 2-OH estrone independently of the volume of sample, and the detection limit was between 100 and 240 ng/L (10-24 pg per tube). The ED50 was 370 ng/L, and inter- and intraassay CVs for low, medium, and high concentrations were 22.5%, 22.8%, and 19.9%, and 17.4%, 14.3%, and 10.8%, respectively. Median concentrations measured in 14 controls and 33 postmenopausal patients with breast cancer were 0.96 and 1.55 micrograms/g creatinine, respectively, but there was considerable overlap between values from controls and patients.