A RIA for 2-hydroxyestrone (2-OHE₁) in rat plasma has been developed. The assay employs an antiserum that is specific for catechol estrogens. Specificity is further ensured by purification of plasma extracts on Sephadex LH-20 columns before RIA. Blood was collected at 0 C in the presence of ascorbic acid to prevent oxidation. Under these conditions, the conversion of 2-OHE₁ to methylated derivatives was found to be negligible. Plasma 2-OHEi, LH, FSH, PRL, estradiol, and progesterone were measured at 3-h intervals throughout the 4-day estrous cycle of the rat. The 2-OHE₁ concentration varied from undetectable to 11 pg/ml plasma. No clearly defined relationship with the other hormones analyzed was observed. Thus, it is unlikely that changes in circulating 2-OHE₁ levels are involved in the regulation of the gonadotropin surge and ovulation.