Vasoactive intestinal polypeptide (VIP) in neocortex affects neuronal excitability as well as cortical blood flow and metabolism. Interneurons immunoreactive for VIP (VIP-IR neurons) are characterized by their predominantly bipolar appearance and the radial orientation of their main dendrites. In order to determine whether the morphology of VIP-IR neurons is related to the functional organization of the cortex into vertical columns, we combined both immunostaining of neurons containing VIP and cytochrome oxidase histochemistry for visualizing barrels, morphological layer IV correlates of functional columns, in the primary somatosensory (barrel) cortex of rats. VIP-IR neurons were localized in supragranular (48%), granular (16%), and infragranular layers (36%) as well as in the white matter. In the granular layer, a clear trend that more neurons were located in interbarrel septa rather than in barrels could be observed, resulting in a neuronal density which was about one-third higher in the septal area. VIP-IR neurons from the different cortical layers were three-dimensionally reconstructed from serial sections by using a computer microscope system. The neurons were mostly bipolar. Striking morphological differences in both axonal and dendritic trees were found between neurons whose cell bodies were located in supragranular, granular, and the upper part of infragranular layers, and those whose cell bodies were located in the area below. The former had dendrites which often reached layer I, where they bifurcated several times, and axonal trees which were particularly oriented vertically, with a tangential extent smaller than the width of barrels. Therefore, these neurons were mostly confined to either a barrel- or septum-related column. By contrast, the dendrites of neurons of the latter group did not reach the granular layer. Furthermore, these neurons had axons with sometimes very long horizontal collaterals, which often spanned two, in one case three, barrel columns. It is proposed that the differential morphology of neurons with different locations as stated above parallels to some extent the divergence of input streaming into the corresponding layer-defined areas. As a possible consequence of this, VIP-IR neurons may be capable of adapting the excitability and metabolism of cortical compartments either in a spatially limited or more extensive way.