Simultaneous determinations of plasma levels of oestradiol-17beta, progesterone and luteinizing hormone (LH) during the ovulatory cycles of hens were performed using radioimmunoassay techniques without chromatography. Blood samples were collected from the wing vein of 7 hens with regular ovulatory cycle at intervals of 1, 2 or 4 h throughout 36 h starting 24-25 h before the second egg (C-2) ovulation of the clutch and ending 11-12 h after C-2 ovulation. The oestradiol-17 beta level of the plasma during the ovulatory cycle varied from 50 pg/ml to 250 pg/ml. The value was high 24 h before ovulation and decreased until 10 h before ovulation, then increased and reached a peak 4-5 h before ovulation, when it started to decrease again until 12 h after ovulation. The plasma levels of progesterone varied from 1 ng/ml to 6 ng/ml, and reached a peak 4-6 h before ovulation. The plasma LH value varied from 1 ng/ml to 4 ng/ml, and the changes in the levels corresponded well with that of progesterone, also showing a coincident peak.