Bone morphogenetic proteins (BMPs) are potent osteoinductive agents for bone tissue engineering. In order to define optimal properties of a novel autologous bone graft substitute (ABGS) containing rhBMP6 within the autologous blood coagulum (ABC) and ceramic particles as a compression resistant matrix (CRM), we explored the influence of their amount, chemical composition and particle size on the quantity and quality of bone formation in the rat subcutaneous assay. Tested ceramic particles included tricalcium phosphate (TCP), hydroxyapatite (HA) and biphasic calcium phosphate ceramic (BCP), containing TCP and HA in 80/20 ratio of different particle sizes (small 74-420 μm, medium 500-1700 μm and large 1000-4000 μm). RhBMP6 was either mixed with ABC or lyophilized on CRM prior to use with ABC. The experiments were terminated on day 21 and implants were analysed by microCT, histology and histomorphometry. Addition of CRM to ABGS containing rhBMP6 in ABC significantly increased the amount of newly formed bone and the optimal CRM/ABC ratio was found to be around 100mg/500 μL. MicroCT analyses revealed that all tested ABGS formulations induced an extensive new bone formation and there were no differences between the two methods of rhBMP6 application as determined by the bone volume. However, the particle size played a significant role in the quantity and quality of bone formed. ABGS containing small particles induced new bone forming a dense trabecular network, cortical bone at the rim, bone and bone marrow in apposition to and in between ceramic particles. ABGS containing medium and large particles also resulted in new bone on the surface of particles as well as inside the pores. Histomorphometrical analysis revealed that the ceramics particle size correlated with the quality of trabecular pattern of newly formed bone, bone/bone marrow ratio as observed in apposition and between particles, and the ratio between the cortical and trabecular bone. By employing rat subcutaneous implant assay, we showed for the first time that the size of synthetic ceramics particles affected the osteogenesis as defined by both the quantity and quality of ectopic bone.Copyright © 2020. Published by Elsevier Inc.