An assay for the quantitative determination of 2-hydroxyestrone and 2-methoxyestrone in human urine is described. The analytical procedure involves several purification steps: XAD-2 column chromatography of urine (1 ml), hot acid hydrolysis under reducing conditions, extraction with benzene/ethyl acetate, separation of monophenolic steroids from catecholestrogens by the formation of borate complexes, and partition between different solvents. Quantitation is achieved by radioimmunoassay using highly specific antibodies. For correction of procedural losses [6, 7-3H2] 2-hydroxyestrone and [6, 7-3H2] 2-methoxyestrone are used as internal standards. The method is highly specific as checked by comparison to a double-isotope-derivative method and a newly developed gas chromatography-mass spectrometry procedure. Using this method the urinary excretion of 2-hydroxyestrone and 2-methoxyestrone was studied in children, men, cycling, pregnant and postmenopausal women. Special interest was focussed on the molar ratios of 2-hydroxyestrone to 2-methoxyestrone and the so called "total estrogens" which vary markedly within the different groups investigated. The excretion of 2-hydroxyestrone is especially favoured in women during the menstrual cycle and pregnancy.