The purpose of this study was to measure bone-regenerative effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) in rat calvarial critical-size defects (CSDs). CSDs (8 mm in diameter) were treated with either 1) 2.2 micrograms rhBMP-2 combined with insoluble collagenous bone matrix (ICBM), 2) 6.5 micrograms rhBMP-2 plus ICBM, 3) ICBM alone, or 4) demineralized bone matrix (DBM), for 7, 14, or 21 days. Multiple linear regression showed that rhBMP-2 had a significant time- and dose-dependent effect on bone regeneration (P < .05). After 7 days, new calcifying cartilage and remineralizing ICBM, with an occasional zone of new woven bone, was evident in defects treated with rhBMP-2/ICBM. By 14 days, both doses of rhBMP-2 reconstituted with ICBM had induced more bone formation than ICBM alone or DBM, and 6.5 micrograms was superior to 2.2 micrograms. There was no evidence of adverse cellular response. This study shows for the first time that rhBMP-2 could restore osseous form to a calvarial defect. In addition, osteoregeneration was accelerated by the higher dose of rhBMP-2.