The repair of bone defects can be induced experimentally with bone morphogenetic protein-2 (BMP-2) producing fat-derived stem cells, but this ex vivo tissue engineering method requires the isolation and long-term culture of autologous cells. To develop an expedited bone repair strategy, we transferred BMP-2 cDNA directly to autologous fat tissue fragments that were held in culture for only 24 h before implantation. We evaluated the ability of such gene-activated fat grafts to regenerate large segmental bone defects in rats. Fat tissue was harvested from 2 of 35 male Fischer 344 rats used for this study. The fat tissue fragments were incubated with an adenoviral vector carrying the cDNA encoding either BMP-2 or green florescent protein (GFP), or they remained unmodified. According to their group, the segmental femoral bone defects of 33 rats were filled press fit with either BMP-2-activated fat tissue, GFP-transduced fat tissue, or unmodified fat tissue. Another control group remained untreated. Femora were evaluated by radiographs, microcomputed tomography, biomechanical torsional testing, and histology. Radiographically and histologically, 100% of the femora treated with BMP-2-activated fat grafts were bridged at 6 weeks after surgery. The femora of this group exceeded the bone volume and the biomechanical stability of intact, contralateral femora. Control defects receiving no treatment, unmodified fat tissue, or GFP-transduced fat were filled with fibrous or adipose tissue, as evaluated by histology. The use of BMP-2 gene-activated fat tissue grafts represents an expedited and effective bone repair strategy that does not require the extraction and expansion of stem cells.