These studies were designed to determine the reliability of in vitro tensile testing to measure the temporal development of regenerate bone strength in rats during limb lengthening (distraction osteogenesis, DO). External fixators were placed on the right tibiae of 36 virus-free, 400-450 g male Sprague Dawley rats, and osteotomies (n = 33) were performed. Distraction was initiated the following morning (0 day latency) at 0.4 mm/day and continued to day 20. The 8 mm gap was allowed to consolidate for up to 50 days (day 70 postop). Contralateral unoperated and operated (fixator only) controls were included. On days 20, 30, 50 and 70 postop, the rats were anesthetized, and their tibiae were radiographed prior to undergoing sacrifice for histological or tensile analysis. On day 70, an additional group was tested by three-point bending. Radiodensity measurements demonstrated progressive mineralization of the DO gap, and histology confirmed typical intramembranous ossification of collagen bundles oriented parallel to the distraction force. Tensile stiffness increased significantly between days 20 and 30 postop, this increase correlated with initial radiographic and histologic bridging of the DO gap. Energy to failure and ultimate tensile strength increased progressively to day 70. At day 70, the force to failure for three-point bending was 65% of control tibiae. In conclusion, in vitro tensile testing provides a reliable method to test the development of structural integrity during the early stages of DO. Therefore, the biomechanical effects of postulated modulators of bone repair can be measured during early stages (bone formation, bridging, early consolidation) of DO in a rat model.