Understanding the basic neuronal building blocks of the neocortex is a necessary first step toward comprehending the composition of cortical circuits. Neocortical layer VI is the most morphologically diverse layer and plays a pivotal role in gating information to the cortex via its feedback connection to the thalamus and other ipsilateral and callosal corticocortical connections. The heterogeneity of function within this layer is presumably linked to its varied morphological composition. However, so far, very few studies have attempted to define cell classes in this layer using unbiased quantitative methodologies. Utilizing the Golgi staining technique along with the Neurolucida software, we recontructed 222 cortical neurons from layer VI of mouse barrel cortex. Morphological analyses were performed by quantifying somatic and dendritic parameters, and, by using principal component and cluster analyses, we quantitatively categorized neurons into six distinct morphological groups. Additional systematic replication on a separate population of neurons yielded similar results, demonstrating the consistency and reliability of our categorization methodology. Subsequent post hoc analyses of dendritic parameters supported our neuronal classification scheme. Characterizing neuronal elements with unbiased quantitative techniques provides a framework for better understanding structure-function relationships within neocortical circuits in general. Indexing terms neocortex layer VI; Golgi; neuronal morphology; barrel cortexThe neocortex is composed of six distinct layers, with each layer containing different morphological phenotypes that presumably subserve different processing roles (Mountcastle, 1997). For example, the excitatory spiny stellate cells and small pyramidal neurons in layer IV receive strong thalamo-cortical input (see Davis and Sterling, 1979;White, 1989). By contrast, the excitatory neurons of the infragranular layers (V and VI) are composed predominantly of larger pyramidal cells (Jones, 1984;White, 1989;Rocco and Brumberg, 2007). Although layer IV is the principal input layer, layers V and VI originate most of the cortical output (Jones, 1984;White, 1989). Within the infragranular layers, layer VI is of particular interest, primarily because of the diverse population of neurons and its complicated microcircuitry. Layer VI receives thalamic and cortical inputs and gives rise to corticothalamic feedback projections as well as corticocortical, corticoclaustral, and commissural fibers (Tombol, 1984;Katz, 1987;