The pathways of biosynthesis of androgens have been explored in vitro in the stromal tissue (interstitium) of normal human ovaries by simultaneous incubation of substrate amounts of δ5-pregnenolone-3H and progesterone- 14C. From the distribution of 3H and 14C in the 8–9 radioactive steroids examined, both the δ4-3-ketosteroid and the δ5-3β-hydroxysteroid pathways appear to be involved in the synthesis of δ4-androstene-3,17-dione, the most prominent steroid formed. Among the 5 ovarian specimens incubated, tissue from a pregnant woman and to a lesser extent, tissue from a patient in the 18th day of the menstrual cycle were the most active; tissues from a menopausal patient and from the follicular phase of the cycle were considerably less active. Estrogen formation appeared to be associated with the outer or cortical portion of the ovary in the single specimen examined this way.