Mechanotransduction in the regulation of cellular responses has been previously studied using elastic hydrogels. Because cells interact only with the surface of biomaterials, we are focusing on the molecular mobility at the outermost surface of biomaterials. In this study, surfaces with the mobile Arg-Gly-Asp-Ser(RGDS) peptide have been constructed. Cell culture substrates were coated with ABA-type block copolymers composed of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) segments (A) and a polyrotaxane (PRX) unit with RGDS bound to a-cyclodextrin (B). Adhesion, morphological changes and actin filament formation of human umbilical vein endothelial cells were reduced on the surfaces containing mobile PRX-RGDS in comparison to the immobile RGDS surfaces constructed from random copolymers with RGDS side groups (Prop-andom-RGDS). In the neurite outgrowth assay using rat adrenal pheochromocytoma cells (PC12), only 20% of adherent PC12 cells had neurites on PRX-RGDS surfaces, but more than 50% did on the Random-RGDS surface. The beating colony of dimethyl-sulfoxide-treated mouse embryonic carcinoma cells (P19CL6) were found 10 and 14 days after induction on PRX-RGDS and Random-RGDS surfaces, respectively. After 22 days, the beating colony disappeared on PRX-RGDS surfaces, but many colonies remained on Random-RGDS surfaces. These data suggest that the molecular mobility of the cell-binding ligand on the outermost surface of materials effectively suppresses the actin filament formation and differentiation of these functional cell lines, and may be used as a culture substrate for immature stem cells or progenitor cells.