Monolayers of third-generation amphiphilic poly(amido amine) (PAMAM) dendrimer were prepared at the air/water interface and then were successfully transferred onto silicon wafers by a horizontal lifting method. A comprehensive characterization of the PAMAM monolayers was made, structurally and dynamically, by scanning force microscopy in conjunction with X-ray reflectivity and X-ray photoelectron spectroscopy. The PAMAM molecules sit on the substrate with an oblate shape, in which hydrophilic core and hydrophobic alkyl end groups were toward substrate and air sides, respectively. In the PAMAM monolayers, distinct three, alpha(a)(-), alphaH(2)O(-) and delta(-), relaxation processes were observed on lateral force-temperature curves. They were assigned to segmental motion, plasticized segmental motion by residual water molecules, and a local mode relaxation, respectively.