Plasma levels of 2-hydroxyestrone (2-OHE1) and 2-hydroxyestradiol (2-OHE2) were determined by a new radioimmunoassay which employed a short Sephadex LH-20 column chromatography for the purification of samples and the antiserum to 2-hydroxyestrone-17-(O-carboxymethyl)oxime-BSA conjugate for assay. The plasma value was below the detection limit for the assay (approximately 15 pg/ml) in men and non-pregnant women, but rose 20-200 pg/ml during pregnancy in 2-OHE1 and around 15 pg/ml in the 3rd trimester of pregnancy in 2-OHE2. There was no significant difference of plasma 2-OHE1 level between normal pregnancy and toxemic pregnancy with hypertension, in the 3rd trimester. The plasma level was very low in all of three subjects with the placental dysfunction in toxemic pregnancy. The plasma metabolic clearance rate (MRCp) of 2-OHE1 and 2-OHE2 were determined in normal adults by two methods; infusion of unlabeled 2-OHE1 and 2-OHE2 to equilibrium with radioimmunoassay of their plasma levels, and infusion of [3H]-2-OHE1 and [3H]-2-OHE2 to equilibrium with measurement of chromatographically purified their tritium. The MCRs by the former and latter methods was 40-70 X 10(3) and 15-50 X 10(3) in 2-OHE1, and 18-29 X 10(3) and 12-14 X 10(3) l/day in 2-OHE2, respectively. The major plasma metabolite comigrated with 2-methoxy compounds to each catecholestrogen. The t1/2 of disappearance rate by the method of infusion of unlabeled compounds was approx. 45 s in 2-OHE1 and 90 s in 2-OHE2. When [3H]-2-OHE1 and [3H]-2-OHE2 were incubated with blood samples of adults, 2-methoxy compounds also rapidly formed. From these results it is concluded that the extremely high MCRp of 2-OHE1 and 2-OHE2 make it unlikely these compounds circulate peripherally except in pregnancy in levels sufficient to produce the physiological effects on estrogen receptors or catecholamines.