The right tibia was broken manually in 56 rats weighing 100 g; the fracture was stabilized with an intramedullary steel wire. Groups of rats were killed after 3-30 days. The fracture with its surrounding musculature was dissected out and immediately frozen to -70 degrees C. Cryostat sections of the fracture region were stained with hematoxylin/eosin, toluidine blue, and immunoenzymatically with collagen II antibodies. Another series of 30 fractured rats were killed after 1-15 days, and the fractures were examined histologically after decalcification with EDTA. Two types of callus were observed. The periosteal-endosteal callus started as proliferation of pre-osteoblasts without inflammatory cells on Day 1 and developed bone trabeculae from Day 3 until Days 8-10, but not thereafter. The cartilaginous callus was formed by condensation of fibroblast-like mesenchymal cells mixed with inflammatory cells outside the periosteal callus and started on Day 5 at the fracture fragments denuded of periosteum. The cells differentiating to cartilage seemed to migrate from the surrounding musculature and its newly formed vessels. The enchondral bone formation started close to the periosteal callus from which vessels were piercing into the then hypertrophic and mineralized chondrocytes on Day 11. We conclude that the bridging callus is formed by fundamentals of periosteal callus derived from predetermined cells and the bridge of enchondrally formed bone trabeculae by cells migrating from outside.