Yolk steroid hormones have been documented to have growth and behavior effects on hatchlings in several avian species. The purpose of these investigations was to determine initial levels of androstenedione (A), dihydrotestosterone (DHT), estradiol (E2), and testosterone (T), and document any changes in those hormones during the course of embryonic development in the Leghorn chicken, Gallus domesticus. Eggs were collected, labeled for hen of origin and egg sequence, incubated at 37.8°C, and sacrificed at predetermined times during development. The embryos were staged, the yolk material collected, homogenized and hormones extracted. A, DHT, E2, and T were separated via column chromatography and hormone levels determined using radioimmunoassays (RIAs). Results indicate a significant decrease in A and T during embryonic development, similar to that reported by our laboratory for the alligator, with A levels being significantly greater initially than levels of all other hormones. Changes in DHT mirrored changes in T levels. Chicken E2 yolk content dynamics differ from those we have measured in both the turtle and the alligator. After an initial decline, E2 in the yolks of chicken eggs undergoes a significant increase at the end of development, between embryonic stages 40 and 45 (days 14 and 20 of development). As the increase is much larger than could be accounted for by hormones present in the yolk material, this may represent early embryonic production of steroid hormones by the developing gonads. J. Exp. Zool. 293:594–600, 2002. © 2002 Wiley-Liss, Inc.