The purpose of this pilot study was to prefabricate a vascularized bone graft by using a vascularized periosteal flap containing osteoprogenitor cells, a structural matrix, and recombinant human bone morphogenetic protein-2 (rhBMP-2). In a rat model, a periosteal flap vascularized by the saphenous artery and vein was dissected off the medial surface of the tibia. This flap consisted of three layers-periosteum, muscle, and fascia-and was tubed on itself to form a watertight chamber that was then transferred on its vascular pedicle to the groin. A total of 78 vascularized periosteal chambers were constructed in 39 animals and divided into 10 groups. In group 1, the periosteal chamber was left empty. Groups 2, 3, and 4 consisted of the periosteal flap and rhBMP-2, but in group 3, the proximal vascular pedicle was ligated, and in group 4, the flap was harvested without the periosteal layer and turned inside out. Groups 5 through 10 consisted of the vascularized periosteal flap containing several different structural matrices (calcium alginate spheres, polylactic acid, or demineralized bone matrix) with or without rhBMP-2. Animals were killed at 2, 4, or 8 weeks in each group. The presence and density of any new bone formation was evaluated both radiologically and histologically. Significant bone formation was seen only in those periosteal flaps containing rhBMP-2 and either the calcium alginate or polylactic acid matrix. New bone formation increased both radiologically and histologically from 2 weeks to 8 weeks only in the periosteal flaps containing the polylactic acid matrix and rhBMP-2. This preliminary study therefore suggests that four factors-blood supply, osteoprogenitor cells in the periosteal layer, a biodegradable matrix, and rhBMP-2-are required for optimal prefabrication of a vascularized bone graft.