Abstract Dehydroepiandrosterone sulfate (DHEAS) determination in biological fluids was carried out by enzymatic hydrolysis and conversion into estrogens [estrone ( E 1) and estradiol ( E 2)] by the multienzyme system of human placental microsomes. The enzymatic complex consists of sulfatase, 3β-hydroxysteroid oxido reductase and 5en→ 4en isomerase which converts DHEAS into androstenedione (A); the latter component is further converted into estrogens by the aromatase. The resulting estrogens were determined from the NADH formed by the transhydrogenation reactions of human placental dehydrogenase. NADH was measured by bioluminescence. As little as 4 pg was assayable by this rapid enzymatic method, with a coefficient of variation of 8%. The results are in good agreement with radioimmunoassay and the method is suitable for routine use.