Calcium homeostasis of in ovo (normal) and ex ovo (shell-less) turkey embryos was investigated at 15, 18, and 21 days of incubation. Hypocalcemia and an elevation in circulating 1,25(OH)2D3 in ex ovo embryos were observed by 15 days. Calcium and phosphorus concentrations in femora and tibiae in ex ovo embryos were significantly lower compared to their normal counterparts. These results suggest that shell calcium mobilization is required prior to 15 days of incubation for maintaining serum calcium and supporting bone mineralization. Furthermore, the elevation of 1,25(OH)2D3 is indicative of a functional calcium homeostatic mechanism responding to the absence of the primary calcium source (eggshell) during the second half of turkey embryonic development.