OBJECTIVES We evaluated the role of delta-5-androstenediol (adiol) and its sulphates in health and endocrine diseases. DESIGN Serum and urine samples from healthy adult men and pre and post-menopausal women were analysed by gas chromatography-mass spectrometry to establish reference values. In patients who were either evaluated or treated for endocrine diseases, sequential serum samples were collected and analysed. PATIENTS Reference values were obtained from 24 healthy male, 23 premenopausal and 30 post-menopausal female volunteers. Adiol and delta-5-androstenediol-3-sulphate (adiol-3S) concentrations were determined in combination with other relevant steroids in patients with either pituitary (n = 5), adrenal (n = 2) or gonadal dysfunction (n = 1), or testicular carcinoma (n = 19). MEASUREMENTS After addition of deuterated adiol as internal standard, serum and urine samples were extracted. Steroid sulphates were hydrolysed. The extracts and hydrolysates were purified on HPLC, adiol was derivatized and finally quantified by gas chromatography-mass spectrometry. RESULTS The calculated reference ranges for adiol and adiol-3S concentrations in serum are respectively: in men 1.78-7.24 and 123-579 nmol/l, in premenopausal women 0.65-6.93 and 21.2-298 nmol/l and in post-menopausal women 0.29-2.90 and 6-1-184 nmol/l. Urinary values varied considerably. In the population with endocrine abnormalities serum adiol and adiol-3S concentrations were compared with other relevant steroids. CONCLUSIONS The wide concentration range of adiol and adiol-3S in urine makes analysis of these steroids in urine of little clinical value. Serum concentrations of adiol and adiol-3S are higher in men than in women. Premenopausal values are higher than post-menopausal. Adiol and adiol-3S in serum are significantly correlated in pre and post-menopausal women, r = 0.51 and r = 0.69 respectively, but not in men. In endocrine patients the serum concentrations of adiol show an ACTH or LH dependency in women; adiol correlates with cortisol, dehydroepiandrosterone or androstenedione and, in males, additionally with testosterone. However, in several situations adiol correlates with none of these steroids. Although adiol secretion can be stimulated by ACTH and LH, the level of serum adiol is also determined by other factors. Finally, in adrenal carcinoma serum adiol and adiol-3S may be used as tumour markers