This study was designed to investigate the responses of bone cells to a deproteinized bovine bone material, Bio-Oss(R) (Geistlich-Pharma, Wolhunsen, Switzerland), which was grafted in artificial bone defects of rat femurs. Standardized bone defects in the cortical bone of the right femurs were grafted with Bio-Oss(R) particles. Narrow penetrations were prepared on the bottom of the cavity, enabling osteogenic cells to migrate from the bone marrow. A defect in the left femur without Bio-Oss(R) was used as a control. The treated femurs were histochemically examined at 1, 3, 5, 7, and 14 days after the operation. At day 1, no osteogenic migration into the cavities occurred in either the control or experimental groups. At day 3, alkaline phosphatase (ALPase) immunohistochemistry showed a migration of the positive cells at the bottom of the cavities of the experimental groups, but not in the control ones. At day 5, new bone formation was recognized at the bottom of the cavity of both groups. In the experimental group, ALPase-positive cells were localized on Bio-Oss(R) and/or on the thin bone matrix that covered this material. The superficial layer of Bio-Oss(R) underlying the newly formed bone exhibited osteocalcin immunoreactivity. Transmission electron microscopy revealed osteoblasts depositing bone matrices - including collagen fibers - on the surface of Bio-Oss(R). At days 7 and 14, woven bone occupied the previous cavities of both control and experimental groups, accompanied by osteoclasts. Thus, Bio-Oss(R) appears to serve as a scaffold for osteogenic cells as well as to promote osteoblastic differentiation and matrix synthesis.