SUMMARY: Two mares were operated on during oestrus; in the first animal a mixture of [16-3H]pregnenolone and [4-14C]androstenedione was injected into the largest Graafian follicle; in the second animal, [4-14C]pregnenolone and [7-3H]androstenedione were injected into the follicle. By cannulating the ovarian veins it was possible to measure the rate of transfer of radioactivity from the follicular fluid into the ovarian vein. This ranged from 3 to 9% of the injected dose per hour. At the end of each experiment, 45–52 min. after the injection of the labelled precursors, the follicular fluid was aspirated. Negligible amounts of radioactivity were recovered from the rest of the ovary and from the 'conjugated' fraction of the ovarian vein plasma and follicular fluid. Within the follicle 6·2 and 12·2% of the injected pregnenolone were converted to progesterone. Further metabolism to 17 α-hydroxyprogesterone, androstenedione and oestradiol-17β was very limited. Oestradiol-17β isolated from the ovarian vein had a higher specific activity than that from the follicular fluid while the 3H: 14C ratios were similar in both compartments. In addition, the 3H:14C ratios of androstenedione and oestradiol-17β in the ovarian vein were similar. Therefore it seems likely that oestradiol-17β in the follicular fluid is derived mainly from an extra-follicular source, probably the theca interna cells.