The adhesion of fibroblast on polymer bilayers composed of a glassy polystyrene (PS) prepared on top of a rubbery polyisoprene (PI) was studied. Since the top PS layer is not build on a glassy, or firm, foundation, the system becomes mechanically unstable with decreasing thickness of the PS layer. When the PS film was thinner than 25 nm, the number of cells adhered to the surface decreased and the cells. could not spread well. On a parallel experiment, the same cell adhesion behavior was observed on plasma-treated PS/PI bilayer films, where in this case, the surface was more hydrophilic than that of the intact films. In addition, the fluorescence microscopic observations revealed that the formation of F-actin filaments in fibroblasts attached to the thicker PS/PI bilayer films was greater than those using the thinner PS/PI bilayer films. On the other hand, the thickness dependence of the cell adhesion behavior was not observed for the PS monolayer films. Taking into account that the amount of adsorbed protein molecules evaluated by a quartz crystal microbalance method was independent of the PS layer thickness of the bilayer films, our results indicate that cells, unlike protein molecules, could sense a mechanical instability of the scaffold.