In excitable tissues, extensive neuronal activity or pathophysiological conditions, such as spreading depression, ischemic infarct, or epileptic seizure, are accompanied by changes in extracellular space volume. Extracellular space volume, in turn, influences neuronal excitability and extracellular ion concentrations and is, therefore, an important parameter of brain activity. Unfortunately, determination of changes in extracellular space by ion-selective microelectrodes is tedious, restricted to one spot in space at a time and limited in time resolution. In this study we present intrinsic optical signals in vitro as a tool to measure relative changes in extracellular space volume in brain slice preparations with two-dimensional spatial and sufficient time resolution. Evidence is given that the intensity of intrinsic optical signals is linearly correlated to the amplitude of extracellular space volume changes. In contrast, the optical signal is poorly correlated to the concomitant increase in extracellular potassium concentration. We conclude that intrinsic optical signals in vitro are a useful tool to measure the spread of changes in extracellular space volume with high resolution in time and space. In combination with the measurement of the extracellular space at one location using ion-selective microelectrodes, it is possible to calibrate the optical signal to percentile alterations of extracellular space volume.