In rodent somatosensory (barrel) cortex input is processed by whisker-related columns before the integrated output is fed into behaviorally-relevant circuits. The layer-specific activation patterns of the rat barrel cortex were examined with a set-up for scanning functional connectivity in brain slices. Flash-induced release of caged-glutamate at a large number of stimulation sites was used in combination with simultaneous field potential recordings from layers II to VI with five electrodes. The field potentials revealed striking differences between the cortical layers. Glutamate-release in layer IV and lower layer III was most effective in evoking excitation in all other cortical layers, whereas field potentials recorded from layer IV itself were caused by stimulation of a very restricted columnar zone only. Field potentials in layers II and III were strongly driven by stimulation in layer IV and less consistently and much weaker by layer V. Layer V was the only lamina capable of responding to stimulation of all other cortical layers, thus displaying the largest input maps. Layer VI possessed functional connectivity intrinsically and with layer V. These data lead us to suggest that thalamic input may be boosted by its main target layer IV to start a sequence of excitation in layer IV, passing to the supragranular layers and finally reaching the infragranular layers. This sequence is likely to be backed-up by other simultaneous steps of transmission including a layer IV-to-V interaction. We proposed that the increasing size of the receptive fields when sampling granular, supragranular and infragranular layers in vivo, might have its functional basis in the laminar interactions described here in an in vitro preparation.