Taking advantage of the deviation of the glass V(z) characteristics induced by a thin layer of cytoplasm on top of a plane glass surface we calculated the P-wave velocity, attenuation, thickness of the cytoplasmic layer, and density for a fixed endothelial cell in culture. For the first time the calculations were done for the full projection surface of a cell within the lateral resolution of the scanning acoustic microscope operated at 1 GHz (ca. 0.8 μm). Thus a 2-dimensional image of the stiffness distribution of a single cell was obtained at this resolution. The thickness values obtained were compared with the surface topography revealed by a light microscopic method (reflection interference microscopy) and gave good coincidence of the values. The coincidence of the thickness values achieved by two independent methods are in favor of the reliability of the parameters calculated from the acoustic images. High P-wave velocities appear in some F-actin accumulations.