Purpose: Recently, interest in chitosan has increased due to its excellent biological properties such as biocompatibility, antibacterial effect, and rapid healing capacity. On the other hand, hydroxyapatite is used as a bone substitute in the fields of orthopedics and dentistry. The hydroxyapatite-chitosan (HA-CS) complex containing hydroxyapatite nanoparticles was developed for synergy of both biomaterials. The objective of this study was to evaluate the effect of hydroxyapatite (HA)-chitosan (CS) membrane on bone regeneration in the rat calvarial defect. Methods: Eight-millimeter critical-sized calvarial defects were created in 70 male Sprague-Dawley rats. The animals were divided into 7 groups of 10 animals and received either 1) chitosan (CS) 100% membrane, 2) hydroxyapatite (HA) 30%/CS 70% membrane, 3) HA 30%/CS 70%, pressed membrane, 4) HA 40%/CS 60% membrane, 5) HA 50%/CS 50% membrane, 6) HA 50%/CS 50%, pressed membrane, or 7) a sham . surgery control. The amount of newly formed bone from the surface of the rat calvarial defects was measured using histomorphometry, following 2- or 8- week healing intervals. Results: Surgical implantation of the HA - CS membrane resulted in enhanced local bone formation at both 2 and 8 weeks compared to the control group. The HA - CS membrane would be significantly more effective than the chitosan membrane in early bone formation. Conclusions: Concerning the advantages of biomaterials, the HA-CS membrane would be an effective biomaterial for regeneration of periodontal bone. Further studies will be required to improve the mechanical properties to develop a more rigid scaffold for the HA-CS membrane.