The columnar organization of the mammalian neocortex is based on radially oriented axon collaterals which precisely link cells from distinct cortical layers. During development, these interlaminar connections are specific from their initial outgrowth: collaterals form only in the target layers and there are no transient axonal collaterals in the nontarget layers. To examine whether positional cues within individual cortical layers regulate the laminar specificity of collateral formation, explants of cells destined for different cortical layers were cultured on membranes prepared from target and nontarget layers. Axonal growth and branching were examined on homogeneous membrane substrates and on alternating stripes of membranes from different layers. Results show that axons branch preferentially on membrane substrates from those layers that they would target in vivo. In addition, when cortical axons were given a choice to grow on membranes from either their target or their nontarget layer, they exhibited a clear preference for the target layers. This indicates that membrane-associated cues confined to individual layers regulate the formation of collaterals of cortical axons and restrict their growth to their target layers. Heat inactivation of membranes from target layers resulted in reduced axonal branching. The same manipulation of membranes from nontarget layers increased axonal branching for one population of cortical neurons. Taken together, these results suggest that membrane-associated molecules confined to individual layers induce and prevent the formation of axon collaterals in distinct populations of cortical neurons. Thus, the expression of layer-specific cues provides important constraints for the remodeling of local circuits during cortical development.A prominent component of the intrinsic circuitry of the neocortex are collateral connections of axons from pyramidal and spiny stellate cells. Within one cortical layer, tangentially oriented collaterals form regular clusters of axonal branches (1-3) and interconnect cells with similar functional specificities (4-6). In the vertical domain, pyramidal neurons have a main axon that projects out of the cortex and makes local connections within some of the 6 cortical layers (7-11) For instance, the axons of pyramidal cells in layers 2Íž3 give rise to tangentially oriented collaterals in layers 2Íž3 and layer 5 that branch profusely within these layers but do not extend into the adjacent layers 4 or 6 (see Fig. 1 A). On the other hand, many pyramidal cells in layer 6 send their efferent axon back to the thalamus. These axons emit collaterals in layer 6, which ascend to branch in the overlaying layers 4 and 3. As axon collaterals of layer 6 cells traverse layer 5 they make very few branches in this layer. Physiological studies in primary visual cortex revealed that this precise pattern of interlaminar connections generates characteristic response properties of cortical neurons (12)(13)(14).During development, collateral clusters within a cortical layer...