Cell adhesive peptide Arg-Gly-Asp (RGD) was immobilized using ProNectin F (PnF) on a nontreated polystyrene petri dish at a PnF density of 20 ng cm −2 , which is sufficient for primary rat hepatocyte immobilization. The density of PnF on the dish affects cell morphology and expression of the differentiated functions within the range of 2–2500 ng cm −2 . An optimal monolayer state with defined cell boundaries and hepatocyte nuclei was formed on a 2500 ng cm −2 PnF-coated petri dish, and the ammonia metabolic function was expressed at as high a level as in the hepatocyte/spheroid. We conclude that 2500 ng cm −2 PnF enhances the morphological stability and expression of liver-specific functions of the hepatocyte.