Cortical spreading depression is a slowly propagating wave of near-complete depolarization of brain cells followed by temporary suppression of neuronal activity. Accumulating evidence indicates that cortical spreading depression underlies the migraine aura and that similar waves promote tissue damage in stroke, trauma, and hemorrhage. Cortical spreading depression is characterized by neuronal swelling, profound elevation of extracellular potassium and glutamate, multiphasic blood flow changes, and drop in tissue oxygen tension. The slow speed of the cortical spreading depression wave implies that it is mediated by diffusion of a chemical substance, yet the identity of this substance and the pathway it follows are unknown. Intercellular spread between gap junction-coupled neurons or glial cells and interstitial diffusion of K⁺ or glutamate have been proposed. Here we use extracellular direct current potential recordings, K⁺-sensitive microelectrodes, and 2-photon imaging with ultrasensitive Ca²⁺ and glutamate fluorescent probes to elucidate the spatiotemporal dynamics of ionic shifts associated with the propagation of cortical spreading depression in the visual cortex of adult living mice. Our data argue against intercellular spread of Ca²⁺ carrying the cortical spreading depression wavefront and are in favor of interstitial K⁺ diffusion, rather than glutamate diffusion, as the leading event in cortical spreading depression.