We tested the hypothesis that 24R,25-dihydroxyvitamin D3 [24R,25-(OH)2D3] is an essential vitamin D metabolite for the development of normal bone integrity and the healing of fractures. The natural 24R,25-(OH)2D3 and its synthetic epimer 24S,25-dihydroxyvitamin D3 [24S,25-(OH)2D3] were tested alone or in combination with 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3], on normal bone development and other related variables of the Ca2+ homeostasis system [serum Ca2+, 25-hydroxyvitamin D3 (25OHD3), 24,25-(OH)2D3, and 1alpha,25-(OH)2D3 levels] in chicks. Mechanical testing of torsional strength was carried out on the femur. 24R,25-(OH)2D3 (80 nmol/kg diet) alone was sufficient for normal bone growth and integrity similar to that achieved by the vitamin D3-replete controls. Next, chicks were fed a 25OHD3-replete diet (75 nmol/kg diet) for 8 days after hatching, and then 25OHD3 was withdrawn to minimize any residual circulating metabolites before the imposition of standardized tibial fractures 14 days later. Vitamin D metabolites were administered for 2 weeks to determine their effects on the mechanical properties of healed tibia. 24S,25-(OH)2D3 combined with 1alpha,25-(OH)2D3 or 1alpha,25-(OH)2D3 alone resulted in poor healing [strength values of 0.158 +/- 0.011 and 0.123 +/- 0.009 Nm (Newton x meter), respectively] compared with that in the 25OHD3-treated control group (0.374 +/- 0.029 Nm). In contrast, the fractured tibia of the birds fed 24R,25-(OH)2D3 in combination with 1alpha,25-(OH)2D3 showed healing equivalent to that in the control group, with strength values of 0.296 +/- 0.043 Nm. These results suggest that when 24R,25-(OH)2D3 is present at normal physiological concentrations, it is an essential vitamin D3 metabolite for both normal bone integrity and healing of fracture in chicks.