Galvanotropism in a DC field represents a natural model for studying physiological mechanism of cell orientation toward direction of its migration. The auto/paracrine cytotransmitter ACh is chemotactic for various cell types. In a DC field, keratinocytes (KCs) move toward the cathode, wherein ACh accumulates due to its charge, suggesting that galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh. To test this hypothesis, the galvanotaxis chamber was assembled on a glass slide, forming a trough bordered by electrodes, in which KCs were exposed for 1 h to a DC field with a constant voltage of 100 mV/mm and a current of ~0.5 mA. These parameters were equivalent to the physiological strength of the DC field detectable near the edge of skin wounds. Keratinocyte orientation toward the cathode was abolished due to inhibition of ACh production by hemicholinium-3 and restored by the exogenous agonist carbachol that also accumulates at the cathodal pole. A time-course immunofluorescence study of the membrane redistribution of nicotinic ACh receptors (nAChRs) in KCs exposed to a DC field revealed rapid relocation to and clustering at the cell leading edge of α7 nAChR subtype. Inactivation of α7 nAChRs with antagonists or small interfering RNAs inhibited galvanotropism. These results suggest that in a DC field, KCs migrating toward the cathode actually move toward the concentration gradient of ACh. Perhaps, accumulation of nAChRs on the cathodal side of a cell allows a focal ion flux and local osmotic swelling required for the leading lamella and lamellipodium extension.