The direct, local, administration of adenovirus carrying human BMP-2 cDNA (Ad.BMP-2) heals critical-sized femoral bone defects in rabbit and rat models. However, the outcome is suboptimal and the technology needs to provide a more reliable and uniform outcome. To this end, we investigated whether the timing of Ad.BMP-2 administration influenced the formation of mineralized tissue within the defect. Critical-sized defects were created in the femora of 28 Sprague-Dawley rats. Animals were injected intralesionally with a single, percutaneous injection of Ad.BMP-2 (4 x 10(8) plaque-forming units) either intraoperatively (day 0) or 24 h (day 1), 5 days or 10 days after surgery. The femora were evaluated 8 weeks after surgery by X-ray, microcomputed tomography, dual-energy X-ray absorptiometry and biomechanical testing. The incidence of radiological union was markedly increased when administration of Ad.BMP-2 was delayed until days 5 and 10, at which point 86% of the defects healed. These time points also provided greater bone mineral content within the defect site and improved the average mechanical strength of the healed bone. Thus, delaying the injection of Ad.BMP-2 until 5 or 10 days after surgery enables a greater percentage of critical-sized, segmental defects to achieve radiological union, producing a repair tissue with enhanced mineralization and greater mechanical strength.