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.