The objective of this study was to evaluate the cellular contribution to the phenomenon of de novo generation of bone tissue induced by the controlled release of bone morphogenetic protein-2 (BMP-2). Gelatin hydrogels (2 mg) incorporating BMP-2 (3 microg) with different water contents were subcutaneously implanted into the back of enhanced green fluorescent protein-chimeric mice to induce the ectopic de novo generation of bone tissue. The hydrogels incorporating BMP-2 could release BMP-2 at different time profiles. When evaluated radiologically and histologically, the ectopic de novo generation of bone tissue was induced by the controlled release of BMP-2 from the hydrogels around the hydrogel-implanted site. The relative percentage number of green fluorescent protein- to osteocalcin-positive cells recruited into the de novo generated bone tissue depended on the BMP-2 release profile. The higher the percentage, the stronger was the de novo generation of bone tissue. These findings indicate that bone marrow-derived osteoblast progenitor cells were recruited from the blood circulation by BMP-2 release and consequently contributed to the ectopic de novo generation of bone tissue. It is conceivable that the local concentration of BMP-2 modifies the recruitment profile of progenitor cells with an osteogenic potential around the release site of BMP-2, resulting in regulated volume of de novo generated bone tissue.