When skin is wounded, keratinocytes from the cut edges of the epidermis migrate over the wounded area to re-epithelialize the wound. It is not clear which cells of the epidermis have the capacity to migrate and contribute to this re-epithelialization: the less differentiated cells of the basal layer, or the more differentiated, involucrin-positive suprabasilar cells. Here we demonstrate that both involucrin-negative and involucrin-positive cells are able to respond to a directional cue for migration with sustained directional migration. When cultured keratinocytes are exposed to a physiologic DC electric field of 100 mV per mm as a cue to guide migration (galvanotaxis) they migrate toward the cathode with equivalent directionality. The involucrin-positive cells, however, display mean migration speeds approximately one half (23.6 microm per h) of the mean rate achieved by involucrin-negative cells (46.5 microm per h). Despite their decreased migration rates, involucrin-positive cells appear to possess an intact mechanism for sensing a directional signal, transducing that signal, and responding with sustained directional migration. Because electric fields are endogenous in skin wounds, it is likely that both the basal, involucrin-negative cells and the involucrin-positive suprabasilar cells respond to this cue with directional migration. The new observation that involucrin-positive cells can indeed migrate suggests that these cells may also contribute to wound re-epithelialization in vivo.