A radioimmunoassay for the quantitation of the sum of free, glucuronidated and urine is described. The method is reliable and accurate. Using this method, urinary excretion of 16 alpha-hydroxyestrone was determined in normal men, premenopausal women, and postmenopausal women. The values were compared to the urinary excretion of estrone and estradiol. In two women, the urinary excretion of the three estrogens was measured in daily samples throughout a normal menstrual cycle. We conclude that 16 alpha-hydroxyestrone is a quantitatively important urinary estrogen. Inclusion of the measurement of 16 alpha-hydroxyestrone should yield a more accurate assessment of estrogen metabolism.