Whisker movement is somatotopically represented in rodent neocortex by electrical activity in clearly defined barrels, which can be visualized in living brain slices. The functional architecture of this part of the cortex can thus be mapped in vitro with respect to its physiological input and compared with its anatomical architecture. The spatial extent of excitation was measured at high temporal resolution by imaging optical signals from voltage-sensitive dye evoked by stimulation of individual barrels in layer 4. The optical signals correlated closely with subthreshold EPSPs recorded simultaneously from excitatory neurons in layer 4 and layer 2/3, respectively. Excitation was initially (<2 msec) limited to the stimulated barrel and subsequently (>3 msec) spread in a columnar manner into layer 2/3 and then subsided in both layers after approximately 50 msec. The lateral extent of the response was limited to the cortical column defined structurally by the barrel in layer 4. Two experimental interventions increased the spread of excitation. First, blocking GABA(A) receptor-mediated synaptic inhibition caused excitation to spread laterally throughout wide regions of layer 2/3 and layer 5 but not into neighboring barrels, suggesting that the local excitatory connections within layer 4 are restricted to single barrels and that inhibitory neurons control spread in supragranular and infragranular layers. Second, NMDA receptor-dependent increase of the spread of excitation was induced by pairing repetitive stimulation of a barrel column with coincident stimulation of layer 2/3 in a neighboring column. Such plasticity in the spatial extent of excitation in a barrel column could underlie changes in cortical map structure induced by alterations of sensory experience.