Cortical spreading depression (SD) is a propagating wave of neuronal and glial depolarization that manifests in several brain disorders. However, the relative contribution of neurons and astrocytes to SD genesis has remained controversial. This is in part due to a lack of utilizing sophisticated experimental methodologies simultaneously to quantify multiple cellular parameters. To address this, we used simultaneous two-photon imaging, intrinsic optical imaging, and electrophysiological recordings to ascertain the changes in cellular processes that are fundamental to both cell types including cell volume, pH, and metabolism during SD propagation. We found that SD was correlated in neurons with robust yet transient increased volume, intracellular acidification, and mitochondrial depolarization. Our data indicated that a propagating large conductance during SD generated neuronal depolarization, which led to both calcium influx triggering metabolic changes and H(+) entry. Notably, astrocytes did not exhibit changes in cell volume, pH, or mitochondrial membrane potentials associated with SD, but they did show alterations induced by changing external [K(+)]. This suggests that astrocytes are not the primary contributor to SD propagation but are instead activated passively by extracellular potassium accumulation. These data support the hypothesis that neurons are the crucial cell type contributing to the pathophysiological responses of SD.