Many types of cells migrate directionally in direct current (DC) electric fields (EFs), a phenomenon termed galvanotaxis or electrotaxis. The directional sensing mechanisms responsible for this response to EFs, however, remain unknown. Exposing cells to an EF causes changes in plasma membrane potentials (V m ). Exploiting the ability of Dictyostelium cells to tolerate drastic V m changes, we investigated the role of V m in electrotaxis and, in parallel, in chemotaxis. We used three independent factors to control V m : extracellular pH, extracellular [K ؉ ], and electroporation. Changes in V m were monitored with microelectrode recording techniques. Depolarized V m was observed under acidic (pH 5.0) and alkaline (pH 9.0) conditions as well as under higher extracellular [K ؉ ] conditions. Electroporation permeabilized the cell membrane and significantly reduced the V m , which gradually recovered over 40 min. We then recorded the electrotactic behaviors of Dictyostelium cells with a defined V m using these three techniques. The directionality (directedness of electrotaxis) was quantified and compared to that of chemotaxis (chemotactic index). We found that a reduced V m significantly impaired electrotaxis without significantly affecting random motility or chemotaxis. We conclude that extracellular pH, [K ؉ ], and electroporation all significantly affected electrotaxis, which appeared to be mediated by the changes in V m . The initial directional sensing mechanisms for electrotaxis therefore differ from those of chemotaxis and may be mediated by changes in resting V m .Cells migrate directionally in response to many extracellular cues including chemical gradients (chemotaxis), topography, mechanical forces (mechanotaxis/durataxis), and electrical fields (EFs) (electrotaxis/galvanotaxis) (1,3,8,15,27). Electric fields have long been suggested to be a candidate directional signal for cell migration in development, wound healing, and regeneration. The mechanisms used by cells to sense the weak direct current (DC) EFs, however, have remained very poorly understood.One of the immediate effects felt by a cell upon exposure to an EF is a change in the cell membrane potentials (V m ). In an EF, the plasma membrane facing the cathode depolarizes while the membrane facing the anode hyperpolarizes (17, 18). It has been proposed that the changes in V m may underlie electrotaxis. In a cell with negligible voltage-gated conductance, the hyperpolarized membrane facing the anode attracts Ca 2ϩ by passive electrochemical diffusion. This side of the cell then contracts, thereby propelling the cell toward the cathode. In a cell with voltage-gated Ca 2ϩ channels, channels near the cathodal (depolarized) side open, thereby allowing Ca 2ϩ influx. Intracellular Ca 2ϩ levels will rise both on the anodal side and on the cathodal side in such a cell. The direction of cell movement in this situation will depend on the balance between the opposing contractile forces (17). The role of V m in electrotaxis has not yet been directly tested.In thi...