A prominent feature of nervous systems is the organization of synapses into discrete layers, or laminae. Such laminae are essential for the spatial segregation of synaptic connections conveying different types of information. A prime example of this is the inner plexiform layer (IPL) of the vertebrate retina, which is subdivided into at least ten sublaminae. Another example gaining prominence is the layered neuropil of the zebrafish optic tectum. Three recent papers have shed light on the extracellular signals that control the precise stratification of pre- and postsynaptic neuronal processes in these two areas. The new studies implicate well-known axon guidance cues, including class 5 and 6 semaphorins in the retina, as well as Slit in the optic tectum. Remarkably, the short-range action of Slit, which is required for neurite stratification, appears to be achieved by anchoring the secreted guidance factor to the basement membrane at the surface of the tectum.