Rough-surfaced substrates made by a variety of methods have been shown to influence osteoblast proliferation and differentiation. The purpose of this study is to confirm the role of surface roughness in promoting osteoblastic differentiation using tissue culture polystyrene as substrate, by excluding factors other than roughness. Immature osteogenic cells derived from fetal rat calvariae were cultured on the plastic cover strips having varied degrees of roughness created by treatment with four kinds of grinding paper of different particle sizes. The proliferation and gene expression of alkaline phosphatase (ALP) and osteocalcin of the calvarial cells increased on the rough-surfaced cover strips. These levels increased in response to the increase in the degree of surface roughness up to 0.8 microm of average roughness and then decreased to the level observed for the smooth surface. These results demonstrate that the surface roughness itself caused increases in osteoblastic proliferation and differentiation in cell cultures.