Laying hens were killed at hourly intervals during the 26-h laying cycle. The 3 largest follicles of the size hierarchy were removed and plasma samples were obtained from the same hens. The follicle walls and the plasma were assayed by RIA for estrogen (E), progesterone (P4), and testosterone (T). The data show that for E and T there is an inverse relationship between follicle size and hormone concentration. There is no such difference for P4. Both E and T show a significant drop immediately after ovulation; P4 does not. Both T and E concentrations rise significantly and synchronously at about 4 and at 8 h after ovulation. The OAAD assay of plasma shows a peak of LH about that time. P4 does not show such a rise in concentration. About 8 to 10 h prior to the next ovulation T concentration in all 3 follicles begins to rise, P4 rises only in the largest follicle and E only in the smallest. All three steroids reach highly significant peaks about 4 to 6 h prior to the next ovulation. Both OAAD and RIA detect plasma LH peaks at about that time. Plasma E and follicle E peak synchronously at 4 to 8 h; T peaks occur at 10 to 12 h after ovulation and are asynchronous with the T follicle peaks. All 3 steroids begin to rise in the plasma about 10 h prior to the next ovulation and all 3 peak together about 4 to 5 h prior to ovulation. The present data do not allow to distinguish between rates of steroid synthesis and their release into the plasma. Whether the steroid peaks occurring in both follicles and plasma shortly before the next ovulation are caused by LH or are the cause of its release remains to be determined.