Subcerebral projection neurons (SCPNs)are a major subtype of principal cells in neocortical layer 5, which project axons to subcortical targets including the pons, superior colliculus and spinal cord. We previously reported that SCPNs formed a periodic columnar structure termed micro columns and that activity dependent c-Fos expression elicited by visual stimulation was similar between SCPNs in the same microcolumns (Maruoka et al., J. Neurosci. 2011). These observations have suggested that the microcolumns behave as functional units in layer 5. Besides SCPNs, layer 5 contains callosal projection neurons (CPNs) and inhibitory neurons. It has not been known whether these cell types have orderly cell configuration similarly to SCPNs. In this study, we conducted three-dimensional analysis to investigate the configuration pattern of all neuronal types in layer 5. SCPNs and CPNs were labeled by retrograde labeling and inhibitory neurons were visualized using somatostatin-cre and parvalbumin-cre mice crossed with loxP-tdTomato mice. After clearing of the fixed brain by the Sca/e or the SeeDB methods, large areas of the cortex were imaged using two-photon microscopy. SCPNs showed thin columnar structures in all cortical areas investigated. On the other hand, CPNs didn't show columnar structures. Inhibitory neurons were very sparse and didn't show specific configuration patterns. These results revealed cell type specificity of the microcolumn organization.