Purpose: The aim of this study was to investigate the in vivo performance, including bone regeneration and biomaterial degradation, of three hydroxyapatite-based bone substitutes at surgically created defects in the cranium of rabbits. Materials and Methods: A bovine-derived hydroxyapatite (BHA), a synthetic hydroxyapatite (SHA), and a nano-hydroxyapatite with collagen I (nHAC) composite were characterized and then placed in calvarial defects in rabbits. After surgery at 6 weeks and 12 weeks, the animals were sacrificed for further microcomputed tomography, histologic, and histomorphometric analyses. The amounts of newly formed bone, residual grafting materials, connective tissues, and tartrate-resistant acid phosphatase staining positive multinucleate cells were evaluated. Results: The three hydroxyapatite-based bone substitutes displayed different surface topography. BHA had the highest amount of newly formed bone at both 6 weeks and 12 weeks, yet no significant difference was found between the three groups at 6 weeks. The amount of residual materials was significantly higher in the SHA group than the other groups at both time points. The amount of connective tissue was highest in BHA. More multinucleated cells were observed in the nHAC group. Conclusion: The three tested bone substitutes are suitable for grafting defects related to early bone healing. BHA had a better impact on bone formation with the prolongation of time. An integration of collagen type I and nano-hydroxyapatite may not improve bone regeneration of pure synthetic hydroxyapatite but helps to modify the degradation rate of pure hydroxyapatite. Keywords: animal experiments, bone regeneration, bone substitute, degradation, hydroxyapatite