To treat bone loss that is induced by disease or wounds, bone grafts are commonly used. In dentistry, guided tissue regeneration is effective in the treatment of periodontal diseases. However, bone resorption after implantation is a major problem with the bone graft and guided tissue regeneration technique. This study examines a cell pellet culture system without exogenous scaffolds for bone regeneration. First, we examined the effect of ascorbic acid on cells. Transmission electron microscopic observation revealed that cells formed a three-dimensional structure of multiple cell layers after 5 weeks of culturing in medium containing 50 microg/ ml ascorbic acid with the medium changed every 7 days. A single cell pellet was produced by centrifuging cells that were gathered from 10 tissue culture dishes. Van Gieson staining and collagen type I immunostaining showed that the pellet contained collagen fibers and cells that adhered to the collagen fibers. Several of these cell pellets were implanted subcutaneously on the backs of nude mice for 6 weeks. Histology and immunohistochemistry results indicated new bone formation, vascular invasion, and insular areas of calcification. Bone tissue was surrounded by osteoblasts. The appearance of new bone formation is similar to that seen in intramembranous ossification. The present pellet system is reliable and might solve problems of bone resorption after implantation.