A combination of darkfield techniques and infrared videomicroscopy was used to measure the intrinsic optical signal (IOS) in slices of adult rat neocortex. The IOS, which reflects changes in light transmittance and scattering, provides a means of studying the spread of neuronal excitation and its modulation with high sensitivity and spatial resolution. The column-like IOS elicited by orthodromic stimulation is in accordance with models of neocortical circuitry. Blockade of synaptic transmission by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphovaleric acid (D-APV) reduced the IOS. The GABAA agonist muscimol and the neuroactive steroid 5 alpha-tetrahydrodeoxy-corticosterone (5 alpha-THDOC) decreased the spread of excitation, whereas the GABAA antagonist bicuculline increased it. The present data suggest that the spatial spread of excitation in different neocortical layers is delimited by GABAergic inhibition mediated by the activation of GABAA receptors.