The effect(s) of 24R,25-dihydroxyvitamin D₃ [24R,25(OH)₂D₃] on fracture healing was studied in a vitamin D-depleted chick model. 24R,25(OH)₂D₃, together with another hormonally active vitamin D metabolite, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], improved bone mechanical strength parameters (torsional strength, angular deformation, and stiffness) and the ash content. The synthetic epimer 24S,25-dihydroxyvitamin D₃ [24S,25(OH)₂D₃] was not as potent as the natural 24R,25(OH)₂D₃. In light of the ability of the fracture-healing callus to discriminate between 24R,25(OH)₂D₃ and 24S,25(OH)₂D₃, a search was initiated in fracture-healing callus tissue for the presence of a specific 24R,25(OH)₂D₃ receptor. No evidence was obtained for a classical nuclear/cytosol receptor for 24R,25(OH)₂D₃ in the fracture-healing callus. A specific receptor/binding protein for 24R,25(OH)₂D₃ was found in the callus membrane fraction, which showed different ligand binding affinities [K_D = 18.3 ± 1.9 nmol/L, B_max = 43.9 ± 6.0 fmol/mg; relative competitive index (RCI) for 24R,25(OH)₂D₃/24S,25(OH)₂D₃/25(OH)D₃/1α,25(OH)₂D₃ = 100/37/401/2.0] compared with the ubiquitous serum vitamin D-binding protein (RCI = 100/99/219/5). Also, a callus membrane-binding protein/receptor for 1α,25(OH)₂D₃ was detected with a K_D = 0.83 ± 0.35 nmol/L and a B_max = 35.5 ± 5.2 fmol/mg. Thus, we have demonstrated a biological role for 24R,25(OH)₂D₃ in fracture healing and described the presence of its receptor/binding protein in a callus membrane fraction.