The effects of varying dosages of 25-hydroxycholecalciferol [25(OH)D3] in commercial diluent injected into the amnion of developing broiler embryos were investigated in a preliminary trial and 2 consecutive main trials. Evaluation of the results of the preliminary trial showed that the in ovo injection of 0.60 micrograms of 25(OH)D3 is capable of increasing its subsequent levels by approximately 3 fold in the circulation of broiler embryos on 19.5 days of incubation. Similarly, results of the 2 main trials showed that the in ovo injection of 0.60 micrograms of 25(OH)D3 significantly improved hatchability on day 21 of incubation and the subsequent performance and bone characteristics of broilers through 28 days of post-hatch age. In conclusion, it is suggested that the in ovo injection of 25(OH)D3 at a 0.60 micrograms dose may be used to effectively improve hatchability, and the performance and bone characteristics of Ross ~ Ross 708 broilers.