A method to separate radiolabelled urinary estrogens by high performance liquid chromatography (HPLC) is described. Estrogen glucuronides were isolated from the urine of women receiving bolus injections of [4-14C]estrone or [4-14C]estradiol by adsorption on Sep-Pak C18 cartridges and subsequent DEAE Sephadex A25 column chromatography. Following enzyme hydrolysis, free estrogens were extracted and concentrated in methanol-water containing ascorbic acid. HPLC was performed either by C18 reversed phase chromatography using different concentrations of acetonitrile with or without tetrahydrofurane in phosphate buffer or methanol-water as mobile phases, or on a Diol column using chloroform-isooctane-n-hexane or isopropanol-isooctane-n-hexane as mobile phases. 3H-labelled estrogens were added as internal standards, and urinary [14C]estriol, [14C]estradiol and [14C]estrone concentrations could be measured with an interassay coefficient of variation less than 5%. Interassay coefficients of variation for [14C]2-hydroxyestriol, [14C]16 alpha-hydroxyestrone, [14C]2-hydroxyestradiol, [14C]2-hydroxyestrone and [14C]2-methoxyestrone were between 5 and 10%, while interassay coefficients of variation for [14C]4-hydroxyestrone was 14.6%. Recovery of the unstable catechol estrogen 2-hydroxyestrone was comparable to the recovery of the other estrogen metabolites, due to the addition of ascorbic acid throughout the different pre-chromatographic steps. Our method is suitable for the separation of the major labelled estrogen metabolites found in human urine following administration of radiolabelled estrone or estradiol.