Urinary 2-hydroxyestrone was quantitatively determined in the course of several normal human pregnancies. The urine was subjected to hot acid hydrolysis and after chromatographic purification, 2-hydroxyestrone was converted into the corresponding phenazine derivative, which was submitted to a final column chromatography and then quantitated by UV-spectrometry. For correction of procedural losses 2-hydroxyestrone-4-14C was used as internal standard. The urinary 2-hydroxyestrone of different subjects varied within a wide range especially at mid-pregnancy between 100 and 2500 μg/24 h. The day-to-day variation of the excretion of 2-hydroxyestrone was mostly less than 30% of the total value of that day, but sometimes could even reach 60%. The investigation of 2-hydroxyestrone and total estrogens at regular intervals throughout several pregnancies, showed that the excretion of 2-hydroxyestrone generally reached a maximum during the second trimester, while the excretion of the total estrogens steadily increased up to parturition. When analyzing the urines of different subjects during the last 4 months of pregnancy, no correlation appeared to exist for the excretions of the total estrogens and of 2-hydroxyestrone.