Objective(s): We investigated the role of various biomaterials on cell viability and in healing of an experimentally induced femoral bone hole model in rats.Materials and Methods: Cell viability and cytotoxicity of gelatin (Gel; 50 µg/µl), chitosan (Chi; 20 µg/µl), hydroxyapatite (HA; 50 µg/µl), nanohydroxyapatite (nHA; 10 µg/µl), three-calcium phosphate (TCP; 50 µg/µl) and strontium carbonate (Sr; 10 µg/µl) were evaluated on hADSCs via MTT assay. In vivo femoral drill-bone hole model was produced in rats that were either left untreated or treated with autograft, Gel, Chi, HA, nHA, TCP and Sr, respectively. The animals were euthanized after 30 days. Their bone holes were evaluated by gross-pathology, histopathology, SEM and radiography. Also, their dry matter, bone ash and mineral density were measured.Results: Both the Gel and Chi showed cytotoxicity, while nHA had no role on cytotoxicity and cell-viability. All the HA, TCP and Sr significantly improved cell viability when compared to controls (p <0.05). Both the Gel and Chi had no role on osteoconduction and osteoinduction. Compared to HA, nHA showed superior role in increasing new bone formation, mineral density and ash (p <0.05). In contrast to HA and nHA, both the TCP and Sr showed superior morphological, radiographical and biochemical properties on bone healing (p <0.05). TCP and Sr showed the most effective osteoconduction and osteoinduction, respectively. In the Sr group, the most mature type of osteons formed.Conclusion: Various biomaterials have different in vivo efficacy during bone regeneration. TCP was found to be the best material for osteoconduction and Sr for osteoinduction.