Total estrogen concentration in hen plasma varied from less than 25 pg/ml to over 600 pg/ml. In laying hens there were 4 peaks of which the first occurred about 1 h before ovulation and the other peaks followed at about 6-hour intervals thereafter. Layers had low estrogen levels at the time ovulation was expected but did not occur. A broody hen (mid-incubation) had very low levels at all times in the day. Nonlaying hens and layers on days of no ovulation had 2 daily peaks. Estrogen levels were higher in long ovarian cycles (time from ovulation to oviposition) than in short cycles. This may be due to a time shift in phase of the estrogen peaks as the sequence (clutch) of eggs is laid or to a gradual failure of the hypothalamo-pituitary system in the production of gonadotrophin.