Anorganic bovine bone matrix (Bio-Oss((R))) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss((R)). In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss((R)) in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were formed in a white rabbit model and then implanted or not (controls) with Bio-Oss((R)) or BMP-2/Bio-Oss((R)). The Bio-Oss((R)) and BMP-2/Bio-Oss((R)) groups had significantly greater new bone areas (expressed as percentages of augmented areas) than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss((R)) group (16.50 +/- 2.87 (n = 6)) had significantly greater new bone areas than the Bio-Oss((R)) group (9.43 +/- 3.73 (n = 6)) at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss((R)) markedly enhances bone regeneration.