In the last decade, the measurement of activity-dependent intrinsic optical signals (IOSs) in excitable tissues has become a useful tool for collecting data about spatial patterns of information processing in mammalian brain and spread of excitation. Although the extent of the IOS correlates well with the extent of electrical excitation, its time course is much slower, suggesting that it does not directly monitor the electrical activity. The aim of this study was to investigate the mechanisms responsible for generation of IOSs. Coronal neocortical brain slices of juvenile rats were electrically stimulated at the border of layer VI and the white matter. The induced columnar-shaped IOSs were recorded using dark-field video microscopy. At corresponding locations, alterations in extracellular K+ concentration and extracellular space (ECS) volume were registered using ion-selective microelectrodes. After stimulation, a transient increase of extracellular K+ concentration up to 10 mM and a transient decrease of ECS volume by approximately 4% could be observed. The comparison of the time courses of these parameters yielded considerable differences between extracellular K+ concentration increase and IOS, but obvious similarities between alterations in ECS volume and IOS. To test the hypothesis that changes in IOS reflect changes in ECS, but not extracellular K+ concentration, we recorded under conditions that are known to prevent activity-induced changes in ECS, i.e., in low Cl- solutions and in the presence of furosemide. Both treatments similarly decreased stimulation-induced IOSs and alterations of ECS. However, the effect of these treatments on changes of extracellular K+ was different and did not correspond to the changes of IOS. We conclude that activity-dependent IOSs in rat neocortical slices measured by near-infrared video microscopy reveal changes in ECS. Furthermore, the pharmacological and ion substitutional experiments make it likely that activity-induced IOSs are attributable to cell swelling via a net KCI uptake and a concomitant water influx.