A method is described for the determination of unconjugated androstenediol (3β, 17β-dihydroxy-5-androstene) in peripheral plasma based on a competitive protein-binding technique. The procedure includes ether extraction and paper chromatography in the Bush A2 system. Sex steroid binding globulin is derived from patients under estrogen therapy suffering from a carcinoma of the prostate. Separation of bound and unbound androstenediol is carried out by ammonium sulfate precipitation. The precision of the method, evaluated by determining the coefficient of variation of analyses of pools of plasma, was 8.5% for plasma of men and 12.3% for plasma of women. Accuracy was satisfactory, the specificity was proved by mass spectrometry. The mean plasma androstenediol concentration in 19 normal men was 109.9 ± 42.6 (sd) ng/100 ml. In 11 normal women the mean was 61.5 ± 22.7 (sd) ng/100 ml. The difference between male and female values was significant. After stimulation with hCG, plasma androstenediol increased to 246 ng/100 ml in men. Thus, the base value was about doubled. This corresponded to the relative increase in plasma testosterone. In hypogonadal men low concentrations of androstenediol were found. The increase after hCG was insufficient. Values are reported for a number of women, suffering from Cushing's syndrome, adrenal carcinoma, idiopathic hirsutism and primary adrenal insufficiency. From these data it was concluded that in the plasma of men the main source of unconjugated androstenediol are the testes. In females, however, the adrenal seems to be the origin of plasma androstenediol.