Spreading depolarizations (SD) are mass depolarizations of neurons and astrocytes that occur spontaneously in acute brain injury and mediate time-dependent lesion growth. Glutamate excitotoxicity has also been extensively studied as a mechanism of neuronal injury, although its relevance to in vivo pathology remains unclear. Here we hypothesized that excitotoxicity in acute lesion development occurs only as a consequence of SD. Using glutamate-sensitive microelectrodes, we found that SD induced by KCl in normal rat cortex elicits increases in extracellular glutamate (11.6±1.3μM) that are synchronous with the onset, sustainment, and resolution of the extracellular direct-current shift of SD. Inhibition of glutamate uptake with d,l-threo-β-benzyloxyaspartate (TBOA, 0.5 and 1mM) significantly prolonged the duration of the direct-current shift (148% and 426%, respectively) and the glutamate increase (167% and 374%, respectively) in a dose-dependent manner (P<0.05). These prolonged events produced significant cortical lesions as indicated by Fluoro-Jade staining (P<0.05), while no lesions were observed after SD in control conditions or after cortical injection of 1mM glutamate (extracellular increase: 243±50.8μM) or 0.5mM TBOA (glutamate increase: 8.5±1.6μM) without SD. We then used an embolic focal ischemia model to determine whether glutamate elevations occur independent of SD in the natural evolution of a cortical lesion. In both the ischemic core and penumbra, glutamate increased only in synchrony with anoxic terminal SD (6.1±1.1μM) and transient SDs (11.8±2.4μM), and not otherwise. Delayed terminal SDs were also observed in two animals at 98 and 150min after ischemic onset and induced similar glutamate elevations. Durations of SDs and glutamate increases were significantly correlated in both normal and ischemic animals (P<0.05). These data suggest that pathologically prolonged SDs are a required mechanism of acute cortical lesion development and that glutamate elevations and the mass electrochemical changes of SD and are merely different facets of the same pathophysiologic process.