Electric fields exceeding 1 V/cm occur during wound healing, morphogenesis, and tumor growth, and such fields have been shown to induce directional migration of a variety of different cells. However, the mechanism by which electric fields direct cell movement is not yet understood, and the effects on vascular endothelial cells are entirely unknown. We demonstrate that cultured bovine aortic endothelial cells migrate toward the cathode of an applied electric field. Time-lapse microscopic imaging shows that the field suppresses protrusive activity from anode-facing surfaces of the cells while stimulating protrusions from surfaces that face the cathode. The threshold for this response is 1–2 V/cm, similar to field strengths measured in vivo. In addition, fluorescence microscopy shows that lamellipodia projecting toward the cathode are rich in actin filaments. Using quantitative image analysis, we show that the electric field induces a transient 80% increase in the amount of filamentous actin in the cell. Comparison of the distribution of F-actin with total protein distribution indicates that F-actin is asymmetrically distributed in the cytoplasm, being selectively enriched toward the cathode. We propose that physiological electric fields direct cell migration by eliciting an intracellular signal that creates new sites for actin assembly in the cathodal cytoplasm.