The adhesion of L929 fibroblast on polymer bilayers composed of a glassy polystyrene (PS) prepared on the top of a rubbery polyisoprene (PI) was examined. Since the top PS layer is not built on a glassy 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 be well spread. Fluorescence microscopic observations revealed that the formation of F-actin filaments in cells attached to the bilayers was more pronounced with a thicker PS film than with a thinner one. On the other hand, the thickness dependence of the cell adhesion was not observed for the PS monolayer films. Taking into account that the amount of adsorbed protein molecules was independent of the PS layer thickness of the bilayer films, our results indicate that cells, unlike protein molecules, could sense mechanical properties derived from the deeper region of the scaffold.