In this study we used an AFM to investigate the cytoskeletal properties of live Dictyostelium discoideum cells by measuring the local stiffness across individual living cells. We have examined differences in elastic properties of polarized and unpolarized AX3 wild type and the mutant DAip1(-) cells, as well as the differences in the front and rear of the cells in relation to organization of the actin cytoskeleton. We found that the average Young's modulus increases upon polarization for the thin regions of the cell and that in polarized cells, the cell front was stiffer than the cell back. We also found that AX3 cells were stiffer than DAip1(-) cells. This finding suggests that actin polymerization is one of the major determinants of cell motility in Dictyostelium. In addition, a thin agarose film was studied as a model system to examine the influence of the substrate of thin materials probed with the AFM.