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Electrophoretic mobilities of Escherichia coli and Staphylococcus aureus were measured in media of different pH values and ionic strengths at 310 K and the results were analyzed via a new mobility formula which was derived on the assumptions of uniform charge distribution in the cell surface layer of finite thickness and ion-penetrability in the layer. E. coli was shown to have a more negatively charged and less soft surface than that of S. aureus. It is suggested that electrophoretic mobility measurement can be used to detect the difference in surface structure between gram-positive and gram-negative bacteria.