Bone remodeling is achieved through the resorptive activity of osteoclasts and the synthetic activity of osteoblast and it continues throughout life. Cement lines are formed by osteoblasts and are recognized between old and new bone. However, the detailed structure of cement lines has still not been clarified. The present study researched the ultrastructure of cement lines in a ferret femur using light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). On LM, many resorption tunnels were recognized in cortical bones. Some osteoclasts in Howship’s lacunae were observed at the top of the resorption tunnel (cutting cone), and osteoblasts forming some layers were recognized behind osteoclasts (filling cone). The layer of osteoid was observed under osteoblasts, and it was slightly eosinophilic on the decalcified specimen. A very thin layer of cement lines was recognized between osteoid and old bone, and it was slightly basophilic on hematoxylin and eosin (HE). On the non-decalcified sections stained with toluidine blue (TB), cement lines showed anochromasia in the non-etched specimen, but stained with TB in the etched one. On TEM, cement lines were recognized between osteoid and old bone, and they were revealed as extracellular matrices of an afibrillar layer, with a thickness of about 2µm. On BSE analysis, cement lines of osteon were observed as 1- 2µm white lines. Conclusively, the present study indicated that cement lines are extracellular matrices of hypercalcified afibrillar layer having 2µm thickness.