Abstract 1. 1. The role of estr-4-ene-3,17-dione (1) as an ovarian estrogen precursor has been examined. 2. 2. Radiochemically pure 3-hydroxyestra-1,3,5(10)-trien-17-one (11) and 1β-hydroxyestr-4-ene-3,17-dione (III) were isolated from an incubation of minced human ovary with [4- 14C]-1 in the presence of a NADPH-generating system. 3. 3. The formation of II confirms Short's suggestion that I, which he had isolated from equine follicular fluid, may serve as an estrogen precursor in the ovary. 4. 4. The simultaneous formation of II and III parallels the situation in preparations of human-term placenta and supports our previous suggestion that the two products may be related biosynthetically. 5. 5. The demonstration of 1β-hydroxylase activity in human ovary provides an explanation of the formation of urinary estrogen artifacts from labile 1β-hydroxy-19-nor steroids during studies of ig-nor steroid metabolism in non-pregnant women.