Three experiments were conducted to evaluate the effect of differences in in ovo amino acid (AA) injection sites in broiler breeder eggs on subsequent hatchability and BW of chicks. In Experiment 1, an AA solution was injected into eggs with 13-mm. or 19-mm, 27-ga. needles. Uninjected eggs served as controls. Hatchability was decreased (P < 0.05) in eggs receiving AA injections with the 19-mm needle in comparison to the control and 13-mm-injected groups. However, BW of chicks increased (P < 0.05) relative to pre-incubational egg weight by AA injection with the 13-mm. needle. In order to evaluate the in ovo location of AA injections from Experiment 1, India ink was injected into eggs in Experiment 2 with a 13-or 19-mm needle. Immediately after injection, the air cell end of the egg was windowed in order to observe effects of injection site. Windowing of eggs was accomplished by removing a piece of the eggshell over the air cell and the underlying membrane at Day 7 of incubation. The amount of injected India ink was higher in the extra-embryonic coelom in eggs treated by both needles, However, the occurrence of India ink in the extra embryonic coelom was higher (P < 0.05) in the group injected with AA solution using a 13-mm needle as compared to that after injection using a 19-mm needle. The observation of India ink in the amniotic cavity was higher (P < 0.05) in the group injected with AA solution using a 19-mm needle rather than that using a 13-mm. needle. In Experiment 3, treatments consisted of control (uninjected eggs) or windowed eggs. Windowed eggs received AA to the chorioallantoic membrane, the yolk, extra-embryonic coelom, or amniotic cavity at Day 7 of incubation. Hatchability was reduced, but chicks hatched when eggs were windowed and when AA were injected into the yolk sac or extra-embryonic coelom. However, chicks did not hatch when AA were administered to the chorioallantoic membrane or into the amniotic cavity. These results suggest that the best AA injection sites in ovo may be the yolk and extra-embryonic coelom.