Estrone sulfate, quantitatively the most important estrogen in plasma, has previously been determined only after hydrolysis and chromatography. An antiserum raised against estrone glucosiduronate-bovine thyroglobulin was found to be suitable for the specific RIA of estrone sulfate both in plasma and urine. Plasma levels were measured after solvent extraction without hydrolysis or chromatography. The mean (+/-SE) was 972 +/- 79 pg/ml (range, 537-1581) in 15 women in the follicular phase, 1806 +/- 160 pg/ml (range, 814-3358) in 15 women in the luteal phase, and 922 +/- 62 pg/ml (range, 461-1238) in 13 men. The urinary excretion of estrone sulfate, measured after simple chromatographic separation, ranged from 0.8-7.9 micrograms/24 h in men and 5.1-18.7 micrograms/24h in nonpregnant women. This was generally one-seventh to one-half the simultaneous estrone glucosiduronate excretion rate. An approximate mean renal clearance of estrone sulfate calculated from the above values was 2.7 ml/min. The low clearance rate is taken to reflect extensive binding of estrone sulfate by plasma proteins. The splanchnic extraction of estrone sulfate measured in 6 patients undergoing hepatic vein catheterization for diagnostic purposes was 29.8 +/- 11.1%, indicating net uptake of this compound by the splanchnic area.